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Alma Mater Studiorum – Università di Bologna

DOTTORATO DI RICERCA IN

Scienze Zootecniche

Ciclo XXVIII

Settore Concorsuale di afferenza: 07/G1 Settore Scientifico disciplinare: AGR 19

TITOLO TESI

Risk Factors affecting carcass and pork quality in pre-slaughter

period

Presentata da: Agnese Arduini

Coordinatore Dottorato Relatore Prof. Giovanni Dinelli Prof. Leonardo Nanni Costa ----------------------------------- --------------------------------------------- Esame finale anno 2016

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Summary Skin damage and meat quality are today very important issues for the whole pig

industry as well as animal welfare too. These contents have infact a ethical and,

above all, important economical implications.

Several studies have just shown that pre-slaughter phases are very critical for the

animal welfare and consequently for final meat quality traits.

In a very specilized country as Italy in heavy pig rearing for DPO productions, which

are the most famous and eaten for their high quality, is foundamental to limit

carcass and meat defects.

To better investigate the real impact of pre-slaughter period and its entity on

carcass apperance and meat quality, a Risk Assessment approach was applied in this

phase to have a widely idea of which are the potential causes of damage and

worsening of carcass and meat and in which moment they occour.

Risk Assessment (RA) consisted in the observation of the potential causes of carcass

lesions occurred during unloading at the slaughterhouse and during driving of

animals to the stunning point. A total of 1680 Italian heavy pigs were examinated

and frequency of pigs’behaviour was recorded and related to carcass damage on,

ham, loin and shoulder/head. The operators’handling was recorded too. It was

found that, the main potential cause of damage was the use of driving devices by

operators.

The effect of pre-slaughter stress was also evaluated on some quality traits of pork.

Twenty-eight pigs of three different breed (Italian large White, Italian Duroc and

Pietrain) were subjected to two different handling manner (rough and genlty) before

stunning and the level of lactate, pro-, macro- and total glycogen were evaluated in

longissimus dorsi and semimembranosus muscles.

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Pigs rough handled before death showed the tendency to presents muscles with

lower level of pro-, macro- and total glycogen and higher level of lactate, with little

differences among breeds within the parameters. These results indicate that the

type of pre-slaughter handling and stress can potentially adversely affects meat

quality and animal welfare.

Before the arrival at the abattoir, transport is well known to be another very

stressful event for pigs which influence skin damage score and meat quality. In this

work data from 3.650 heavy pig batches were collected to identify the relationship

between the transport and ham aesthetical and technological defects that make

them being rejected from DPO Parma Consortium. The effect of journey duration

and season were related to the incidence of haematomas, lacerations,

microhaemorrhages and veinig defects.

The results shown that short (<37 km) or long (>170 km) travel distances may have

adeverse effects on the incidence of defects on fresh hams together the season of

transport, where Autumn and Spingtime were found to be the seasons with highest

incidence of ham defects.

This thesis confirms how ante-mortem handling is a very important factor not only

for the welfare of pigs but also for the quality of carcass, and raw hams and for the

final meat quality. Risk Assessment could represent a valid tool to monitor pre-

slaughter handling under commercial conditions.

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Table of contents

SUMMARY……………………………………………………………….………………………...…..……………….2

GENERAL INTRODUCTION………………………………………………………………………………………..6

Heavy pigs…………………………………………………………………………………………………………….6

DPO productions and meat quality……………………………………………………………………….7

PRE-SLAUGHTER TREATMENT………………………………………………………………………………..10

Loading…………………………………………………………………………………………………………………11

Transport……………………………………………………………………………………………………………..12

Unloading…………………………………………………………………………………………………………….14

Lairage……………………………………………………………………………………………………………….…16

Stunning……………………………………………………………………………………………………………….19

CARCASS AND PORK QUALITY…………………………………………………………………………………20

Skin bruises………………………………………………………………………………………………………….20

pH………………………………………………………………………………………………………………………..22

DFD and PSE defects…………………………………………………………………………………………….22

Colour………………………………………………………………………………………………………………….24

Water holding capacity…………………………………………………………………………………………25

Drip loss……………………………………………………………………………………………………………….25

Cooking loss………………………………………………………………………………………………………….26

Flavour………..……………………………………………………………………………………………………….27

Tenderness…………………………………………………………………………………………………………..27

AIM……………………………………………………………………………………………………………………..…29

CHAPTER 1:

Risk characterization for carcass lesions during the pre-slaughter steps at unloading

and stunning of Italian heavy pigs……………………………………………….………………………...30

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CHAPTER 2:

Effect of transport distance and season on some defects of fresh hams destined for DPO production……………………………………………………………………………………………………..51

CHAPTER 3:

The effect of the stress immediately prior to stunning in different breed on pro-, macroglycogen, lactate and pork meat quality traits……………………………………………..69

GENERAL CONCLUSIONS……………………………………………………………………………………..…84

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General Introduction

The Italian pig commerce takes a key role in the national livestock and agriculture

production. In the food industry, production of fresh pork and sausages accounts

alone for 45% of meat industry sales (ISMEA, 2008). The national production is

highly focused and specialized on heavy pigs for processing (95% of slaughtering’s)

In particular, this type of rearing is aimed at DPO and GPI products representing

more than 50% of the total value generated by the farms and about 25% of sales

value generated by the meat industry (ISMEA, 2014). Denomination of Protected

Origin (DPO) and Geographical Protected Indication (GPI) are brands which protects

and enhances the quality of products offering to consumers a guaranteed

purchases. This guaranty is assured by the stricted rules included in the respective

production disciplinaries, one for each product. DPO and GPI indicates foods whose

peculiar characteristics depend essentially from the territorial area in which they are

exclusively produced and processed.

Parma and San Daniele hams, the symbol of the Made in Italy food worldwide,

derive from this unique and of great quality and cultural value supply chain. The

breeding of pigs for DPO supply chains is a large part of the Italian pig production

and is concentrated in a fairly small area, where Piemonte, Lombardia, Veneto and

Emilia Romagna regions represent the core, with almost 4000 farms corresponding

to 89, 25% of the total (Regione Lombardia, 2014).The Italian province with the

highest concentration of pigs is Mantova, where for each inhabitant there are on

average four pigs.

Heavy pig

The type of heavy pigs is an Italian characteristic aimed to dry cured ham

production, whose preparation requires thighs coming from pigs reared and

slaughtered in specific areas and with quality characteristics indicated by the

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production regulations of the different Consortia. Heavy pigs intensively reared

accounts for 30-35% of all herds and is concentrated in the Po Valley.

Pig breeds allowed to this production are principally Italian Large White, Italian

Landrace and Duroc, as indicated in the Disciplinary of ham Production and other

DPO pig products and reported by the Italian Herd Book for heavy pigs.

Heavy pigs are slaughtered at a minimum age of 9 months and an average live

weight of 160 kg and more (Parma Ham Disciplinary,1992) in order to achieve

carcasses with a good degree of marbling, large thighs with characteristics suitable

for the processing and transformation into dry-cured ham or other meat products.

The processing industry requires fresh cuts characterized by a narrow range of

weight and subcutaneous fat contents and with a high level of intramuscular fat to

improve uniformity and quality of the end products (Piao et al., 2004).

Ham, especially DPO, is the product with highest commercial value of the Italian

food industry and for this reason the thighs represent 55% of the commercial value

of the heavy pig carcass, even though they represent only 18-20% of carcass total

weight (Pastrello, 2011, Russo, 1990, Chizzolini et al., 1995).

DPO productions and meat quality

Parma ham is the Italian DPO product best known in the world and represents,

alone, 90% of Italian DPO market with a production of almost 9 million hams

branded in 2014.

The raw material, i.e. fresh legs, comes from heavy pigs reared in a limited area, in

accordance with the Regulations of Parma Ham Consortium.

Factors as genetic type, sex, breeding, feeding and slaughter live weight allowed for

such production are clearly reported in the Regulations to get a product with

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specific properties and a meat suitable for processing and transformation (Regione

Lombardia, 2014).

All these aspects together with farming technologies and pre-slaughter treatment

strongly influence the quality of the meat and the characteristics of the thighs

(Rosenvold and Andersen, 2003).

The genetic type influences the chemical, technological and sensory properties of

meat and derived products (Moretti et al., 2009). Over the years, the selection of

breeds and hybrids destined to processing industries lead to a type of heavy pig

which presents evident differences with the pigs slaughtered at 90-110 Kg live

weight. Italian genetic breeding for the heavy pig is focused on high processing

suitability and a fat covered thighs for seasoning, while around the world pigs are

selected and characterized for the high percentage of lean cuts.

Sex and live weight affect both the characteristics of the carcass and meat: castrated

females and males show heavier slaughter weight than intere females (Latorre et al.,

2003, 2004) with an increase in carcass yield. Moreover in castrated females

increases the fatness of primal cuts (loin+shoulder+ham) facilitating the drying and

ripening process of the meat (Candek-Potokar et al., 2002) and improving the

quality of dry-cured products for aroma and flavor (Ruiz-Carrascal et al., 2000;

Banòn et al., 2003). Sex could influence other meat characteristics as reported by

Candek-Potokar et al., 2002 who observed that hams from females had firmer

texture than hams for castrated males and by Nold et al.( 1999) and Latorre et al.

(2003) who highlighted that barrows have lighter and more red colour compared

with gilts and boars are leaner than barrows and gilts for the lower rate of

intramuscular fat (Latorre et al., 2003). Differences in marbling between gilts and

barrows were found by other authors (Furman et al., 2007).

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The effects of sex and market weight on meat quality was demonstrated including

also pH, drip loss,cooking loss, share force, juiceness and overall taste as underlined

by Piao et al. (2004).

A recent study shows how the different rearing technologies are able to influence

the chemical and sensory characteristics of pork (Bonneau and Lebret 2010). The

space available to animals and the presence of straw bedding can lead to an

increase in intramuscular fat with a positive effect on the flavour and juiciness

(Lebret, 2008) and a reduction of cooking loss (Mandell et al., 2006, Matthews at al.,

2001). In addition, the outdoor rearing system seems to influence the characteristics

of carcass and intramuscular fat in meat, in relation to the climatic conditions

(Edwards et al., 2005).

Feeding composition in protein, fatty acids and minerals content including

integration in vitamins and antioxidants influence greatly the quality of the meat

and derived products (Corino et al., 1999; Dugan et al., 2004; Rossi et al., 2010).

Fatty acid composition influences several aspects of meat quality, including tissue

firmness, shelf life and eating quality, particularly flavour (Teye et al. 2006, Isabel et

al., 2003, Wood et al., 2003). Intramuscular fat content can be increased by feeding

pigs protein/lysine-deficient diets in the growing or finishing phases (Teye et al.

2006, Castell et al., 1994, Cisneros et al., 1996 and Wood et al., 2004).

Also the pre-slaughter treatments such as the duration of journey, the density inside

the vehicle and the management of the animals from the farm to the

slaughterhouse, can affect pig welfare and pork traits. Pre-slaughter handling

includes a wide range of different physically and psychologically stressful procedures

(Grandin,1997) that could result in alteration of muscles metabolism and

consequently in a worsening of meat quality (Muchenje, 2011).

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Pre-slaughter treatment

After a rearing period of several months under specific housing conditions, pigs are

transported on road from the farm to the slaughterhouse. The handling of animals

for slaughter includes the exposition of pigs to novel procedures such as loading,

transport, unloading at the abattoir, lairage and driving to the point where they are

stunned. The combination of these stressful situations concentrated in a very short

period of time may have a large effect on the welfare of pigs. For example, fear

reaction can make handling difficult and causes potentially dangerous situation for

animals (Driessen et al., 2013). Physical exercise of animals during the loading of the

vehicle, the moving from a familiar environment into a novel one, the closed contact

with stock people and type of handling have an important impact on animal stress,

welfare, and meat quality. The high level of stress reached by pigs before death

could increases physiological levels of blood cortisol, lactate, CPK and adrenaline

(Troeger 1989; Støier et al. 2001; Hambrecht et al. 2005), heart rate and body

temperature (Schaefer et al. 1989; Griot et al. 2000) whose may lead to defects such

as PSE (pale, soft, exudative) or DFD (dark, firm, dry) meat (Kauffman et al., 1978).

Ante-mortem conditions affects also skin damage score (Faucitano, 2001).

All these factors could represent a commercial problem for swine industry for the

possible financial losses and as well as an ethical concern because can seriously

compromise pigs’ welfare too.

In order to ensure the welfare of pigs in pre-slaughter period, reducing stress and

consequently increasing meat quality, there is the need to invest in new handling

techniques, appropriate facilities (Goettems, 2011) and in training of all operators.

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Loading

The transfer from the familiar pen to the interior of the trailer, associated with the

physical effort induced by the coercion to walk through sloped ramps, make pigs

nervous and more difficult to handle (Driessen 2013, Faucitano 2001).

Moving animals in small group sizes (Lewis and McGlone, 2007) and use appropriate

devices as solid board (Hemsworth, 2000; Correa et al., 2010) help to better a

handling and reduce stress in pigs. Unfortunately, the wrong habit of handling too

many animals to the loading ramp of the truck is widely diffused in the conviction to

accelerate the loading times but getting the opposite result. Due to the coercion of

novelty balk, pigs try to escape or overcrowd to find protection into the group.

Animals hesitation induces also stock people to an excessive use of driving tools,

especially at the entrance of the loading ramp, to move quickly on pigs.

Although a common practice, rough handling should be avoided for its detrimental

effects on animal welfare: it amplified stress in pigs due to fear and agitation.

Exceeding in using ectrical prods has been related to increased heart rate, higher

levels of lactate, incidence of fatigued pigs and resulted in poorer meat quality too

(Weschenfelder, 2013). Prods or sticks has been demonstrated contribute to

increase skin damage score (Faucitano, 2001) and incidence of PSE pork (Driessen et

al., 2013).

The design of loading facilities is another factor that influence animal welfare and

meat quality. In order to drive pigs calmly without panic, slips and falls, there is a

need that ramps and trailers have no slippery floor. Moreover, ramps’ inclination

shouldn’t be above 30°, avoiding excessive physical effort. A hydraulic lift has been

recommended than ramps because it makes the loading operation easier and

quicklier, reducing the need to constraint pigs (Driessen et al., 2013).

To maintain the order during moving is also better not mixing unfamiliar pigs coming

from different pens or batchs . In fact, mixing pigs is often made at loading to fill at

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maximum capacity the decks of lorry (Faucitano, 2001, Gispert et al., 2000). Mixing

pig leads to animal fighting , especially during lairage time, with an increase of

stress and skin damage (Aaslyng et al., 2013).

Transport

The conditions under which slaughter pigs are transported represent a critical point

in pre-slaughter period because can seriously affect pigs’ welfare as well as carcass

damage score.

During transport, pigs are exposed to several stressful situations, such as unfamiliar

and loud noises, new smells, vibrations, lower individual space besides sudden

speed changes of the truck and a frequent contact with handlers (Grandin, 1997).

Vehicle design, placement and microclimate inside the vehicle, season, stocking

density, duration of the journey and driving conditions are the main factors reported

to affect animal welfare and/or the quality meat.

The driving style of the trucker influences the behaviour of the pigs during transport.

When driving is rough animals are subjected to rapid movements i.e. sharp

accelerations and stopping which may cause slips and topples of pigs resulting in

bruising, muscular fatigue, fear (Driessen et al., 2013, Randall et al., 1995). The

physical effort to remain standing, in order to cope with the high level of vibrations

result in higher skin damage score and DFD pork (Barton-Gade et al.,1996b) as

standing pigs are more prone to falling or trampling (Dalla Costa et al., 2007, Barton-

Gade et al., 1996b). A better driving allows pigs to adapt the high level of vibrations

with the standing or lying position (Driessen et al., 2013).

Randall et al., 1996 found that also vehicle type and and the animal location inside

(Barton Gade et al., 1996b, Guise & Penny 1989) are also important both animal

welfare and carcass quality: a large body fixed truck assure a better comfort level

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and the front and rear compartment have a significant effect on meat quality and

carcass blemishes (Dalla Costa et al., 2007). In addition, the deck of the trailer is

important for bruising issue too because pigs transported in the lower deck show

higher damage scores in the middle and shoulder than pigs transported on the

upper deck (Faucitano, 2001, Barton-Gade et al. 1996a).

Loading density in the transport vehicle is an important factor in pre-slaughter stage

contributing to variation in presence of carcass damage and pigs’ welfare in relation

to environmental temperatures.

EU directive 95/29/EC rules European densities of transport fixed at 250 kg/m2 or

0.44 m2/100 kg for normal slaughter pigs of 90-100 kg live-weight (Warriss, 1998) to

ensure animals the space to lie down in their natural position. Regulation CE

N.1/2005 also highlighted this loading density threshold. Out of Europe, this

stocking density is rarely applied in commercial condition as space allowances are

frequently adjusted according to different transport conditions (Faucitano, 2001)

and economical pressures (Warriss, 1998) with real densities ranging from 0.35 to

0.50 m2/100 kg (Weschenfelder, 2013). Nevertheless, there are some evidences

that at low stock densities pigs can change behaviour leading to greater skin damage

incidences (Faucitano, 2001). The negative effects of high densities on meat quality

can be observed alone or combined with other effects, as reported by Carr et al.

(2008) who found that pigs being handled roughly at loading and transported at high

density produced darker pork for lower L* values (Weschenfelder, 2013).

In general, the journey duration could exacerbate the stress of transport in pigs with

a worsening of welfare condition. A longer travel time increases fatigue symptoms

such as tremors, hyperventilation and erythema, as well as incidence and degree of

skin bruising (Mota-Rojas et al., 2006). Gallo et al., 2001 found that after longer

journey more animals show lesions. Long distance transportation results also in

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depletion of energy for prolonged stress of pigs leading to darker carcasses (DFD

meat) and a reduced carcass yield (Mota-Rojas et al., 2006). Controversially, some

authors have demonstrated that pigs transported for short distances failed in

recovering from loading and transport stress producing PSE meat (Sheeren et al.,

2014, Pèrez et al., 2002) more difficult to handle (Grandin, 1994).

Besides transport distances are associated with greater mortality. Barton-Gade et al.

(2007) and Warris (1998) reported that deaths are more frequent on longer

journeys, Werner et al. (2007) affirmed that travel distances longer than 8 h and

shorter than 1 h, both, negatively affected animal welfare with increased mortality

rates. In any case pigs transport carried out with a normal truck shall not exceed 8 h,

but can be prolonged up to 24 h with a special vehicle provided with adequate

ventilation system (Brown et al., 1999) and with continuous access to water during

the journey (Bench et al., 2008; Reg. 1/2005 CE).

The duration of transport is however conditioned by the season because external

temperature is an additional factor affecting pigs welfare during transportation. In

hotter months was observed a reduction of meat quality as a consequence of heat-

stress that involves fatigued pigs (Mota-Rojas et al. 2006). Correa et al. (2013) and

other authors showed a higher risk of PSE pork in summer and DFD in winter. In this

latter season there is evidence of greater frequency of skin damages (blemishes,

haematomas and blood splashing), probably caused by slips and falls for slippery icy

floor (Schereen et al, 2014, Gozlavez et al., 2006). Transport duration and

temperature were found to affect body weight, with the highest weight losses

apparent after 24 h of transport at 35°C (Lewis et al., 2005; Berry & Lewis, 2001a).

Unloading

At the slaughterhouse, pigs should be unloaded as soon as possible (AAFC, 1993) to

avoid temperature stress and its negative consequences on meat quality and animal

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welfare: an increase in carcass bruises and in incidence of exudative pork for

unloading times longer than 30 min was found by Weschenfelder (2013) and

Driessen & Geers (2001). If delay may unavoidable, animals have to be provided

with adequate ventilation (Szent István University, 2009).

Even if is considered less stressful than loading, unloading represents a source of

stress due to novel environment and handling that may cause fear (Dalmau et al.,

2009). The physical effort lead to an increase of heart rates and of plasmatic cortisol

and creatine kinase concentrartion (Geverink et al., 1998, Kim et al., 2004, Brown

et al., 2005). Problems can be caused by the lack of sheltered quays when animals

are subjected to wind, rain and sunlight they balk and may refuse to exit the lorry.

The reluctance of pigs to go forward can be also caused by poor lighting and

inappropriate design and location of the unloading area. Different colours and

shadows may frighten the animals and they preferably walk form a dark to a lighter

place (Driessen et al., 2013). The use of a hydraulic lift to offload allows a gradual

emptying of the truck by compartment using paddles or boards only, increasing the

easiness of handling and reducing the time of procedures (Weschenfelder, 2013,

Jones, 1999). The unloading space must be structured in order to driving pigs

straight to lairage without any obstacles as bottlenecks or corners, that could

damage the skin during the passage. The condition of the surrounding environment

play a key role to for pigs’ responses (Faucitano, 2001) and meat quality (Van de

Perre et al., 2010) as well as the quality of management by operators.

Several studies (Driessen et al., 2013, Rabaste et al., 2007) reported that pigs being

handled gently at unloading were less stressed and adapted better to the lairage

pen environment than pigs being handled with electric prods. Frequent use of

electric prods results in fear and stress, making pig driving more difficult due to an

increase of mounting, slipping and turning (Rabaste et al., 2007) and it is

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responsible for considerable increases in skin damage on the carcasses (Rabaste et

al., 2007, Geverink et al., 1996). Damage to the surface of the carcass after dehairing

is a serious commercial problem, since it downgrading the value of the carcass and

adding costs to remove blemished tissue and reducing the speed chain (Faucitano,

2001).

Lairage

Among the pre-slaughter factors herein examined, lairage was the most important

source of variation determining meat quality.

Resting time at abattoir provides an opportunity for animals to recover from stress

and fatigue of transport and unloading (Warriss, 1987), limiting the presence of

defects in carcass and meat (Faucitano and Geverinck, 2008, Warriss, 2003).

Furthmore resting creates a reservoir of animals aimed at maintaining the constant

speed of the slaughter line.

There is evidence that a lairage time of 1-3 h is optimal and recommended to

recover from stress prior to arrival at the slaughter plant (Pannella-Riera et al. 2012,

Lammens et al. 2007) and to have muscle temperature and ultimate pH in a good

range for meat quality (Zhen et al., 2013). Several studies shown that short lairage

durations resulted in pigs with higher blood level of cortisol, lactate and CK (Salajpal

et al., 2005, Saco et al., 2003). No resting times lead to high incidence of blood

glucose levels (Zhen et al., 2013) and PSE meat (Fortin, 1989; Eikelenboom et al.,

1991). In contrast, longer lairage time provoked greater blood levels of acute phase

proteins (Saco et al., 2003) and fighting (Nanni Costa et al., 2002) associated to

increased skin damage scores (Guàrdia et al., 2009, Nanni Costa et al., 2002) and a

higher risk of DFD pork (Guàrdia et al., 2005). Moreover, Warriss et al. (1998a)

observed a significant reduction of carcass weight and backfat thickness after

overnight lairage. Pigs held in lairage the night before slaughter exhibited skin

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damage in all carcass almost tripled compared with shorter lairage and a little

tendency to DFD condition (Nanni Costa et al., 2002). Overnight lairage leads also to

a depletion of glycogen and a decrease of pH at 1.5 h, colour score and drip loss as

well as an increase of ultimate pH. Lairage time had a significant effect on the

veining defect. Prolonged lairage time increased the presence of the visible vein

network defect on the medial side of the ham (Zgur, 2014). In practice, the resting

times applied are varying from 1 to 15 hours depending on the abattoir size,

availability of pigs for slaughter, transport time, handling procedures and

environmental conditions (Gispert et al., 2000).

Handling, facilities design, environment conditions and mixing are other aspects of

lairage which influence both welfare and meat quality.

Adequate space allowance in resting pens reduce aggressive encounters as reported

by Rabaste et al. (2007) who observed that larger groups pigs in the holding pen

spent more time standing, fighting and were more involved in behaviours as bites

and head knocks than pigs kept in smaller groups. Stress and aggressiveness are also

linked to the time of withdrawal (Nanni Costa et al., 2002). Optimal feed withdrawal

times are suggested to be in between 16 to 24 hours (Eikelenboom et al. 1991) or 12

to 18 hours. If feed deprivation is too long (e.g. with overnight lairage), energy

reserves are empty and there is not enough glycogen to assure a sufficient pH

decline increasing the risk of DFD (Gispert et al. 2000).

Even if the mixing of unfamiliar pigs in lairage is a very common practice, it induces

high levels of aggression among pen mates (Faucitano, 2001, Guise and Penny 1989,

Warriss 1996, Ekkel et al. 1997), which increases skin damage and promotes the

development of PSE/DFD meat (Brown et al., 1999; Gispert et al., 2000). To reduce

the negative impact of this practice on welfare and skin damage, large pens should

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be equipped with mobile dividers in order to keep pigs in small batches avoiding

mixing (Faucitano, 2001, Barton-Gade et al. 1992).

Despite the role of lairage as well as the relevance of resting area that allows

animals to recover from transport stress (Faucitanto, 2001), temperature at lairage

would also represent an important risk factor for animal welfare and meat quality

traits (Lammens et al., 2007). Under extreme temperature conditions (> 30ºC and

RH > 80%), pigs have great difficulty in losing heat and show signs of stress, such as

respiration rate and panting (Santos et al., 1997) with a consequent high lactate and

CPK levels in the blood and a more than two-fold higher incidence of PSE meat

(Warriss et al., 1994). The lack of environmental control may lead to further

economic losses due to death.

A method to improve pigs’welfare in extreme heat conditions, especially during hot

weather, is showering pigs for some minutes in order to reduce the body’s surface

temperature of 3-4 ºC. However, intermittent and frequent showers are

detrimental, as they prevent pigs from resting and lying down (Stzen Istavàn

University). A quick shower during overnight lairage may be needed to avoid skin

reddening (Stzen Istavàn University). At temperatures below 5ºC, showering could

cause animal shivering and may lead to darker pork (DFD) due to muscle energy

depletion to maintain constant the body temperature (Knowles et al., 1998).

Showering provided some minutes before slaughter decreases muscle temperature

and may lead to a better meat quality. Showering pigs in lairage also reduces

aggressive behaviour and facilitates pigs handling upon the entrance into the

stunning chute (Weeding et al., 1993), besides increases electrical stunning

efficiency resulting in an easy and rapid loss of consciousness prior to slaughter

(Wotton, 1996).

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Stunning

During conduction to stunning, animals could be very stressed, due to the fast

handling in small group combined with poorly designed handling systems and rough

procedures. All these factors are detrimental for animal welfare, especially the

excessive use of moving devices such as electric prods that, contrary to the function

which it is used, generates panic among animals and induces mounting, back-up

activity and more fatigued pigs, with delays in the slaughter speed. A worsen meat

quality is also attributed to the use of this device for the higher incidence of bruised

carcasses and PSE pork (Benjamin et al. 2001, Rabaste et al. 2007).

Pre-stunning facilities with inadequate designed could represent a source of skin

damages too. The most critical areas are the passageways from the lairage pens

until the entrance of single chute and the stunning race. In these steps, the close

interaction of animals with handlers make pigs fearful with tendency to

overcrowding, jamming, mounting and high vocalization (Faucitano, 2001). The

abattoirs should be equipped with straight alleys and a minimal number of turns and

corners to encourage the forward movement of pigs. An automatic push gates may

help the staff to move animals, reducing interaction with the human and avoiding

the use of electrical prods.

The design of race feeding the restraining conveyor has been shown to influence the

amount of skin blemishes. An excessive width leads to frequent jamming against

the race walls, resulting also in strong prodding to recreate the order and the speed

chain (Faucitano, 2001). The intense and increasing noise of this point affects pigs’

calm and consequently the handling procedure. Geverink et al. (1998) reported that

the noise produced by the machinery, pressure hoses and pig and human

vocalisations represented a source of stress which evoked huddling and escape

behaviour inducing operators in using sticks (Fraqueza et al. 1998; Grandin 1998).

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A massive use of electric goads is typical of the entrance into the stunning chute for

separating pigs and driving them singly into the stun tunnel at constant speed. The

electric goading and the limited space induce pigs climbing over the backs of other

pen mates in search of protection within the group (Guise and Penny 1989;

Lambooij and Engel 1991), slipping, falling (Dokmanovic et al., 2014) increasing skin

bruises on the carcass, heart rate and negative changes in blood parameters and

affecting post-mortem meat quality (Thorell, 2009). Grandin (2013) recommends

stiff scrub brushes on the end of a stick as an electric prod alternative for moving

pigs up a single-file race, Driessen et al. (2013) affirmed that when a restrainer

conveyor is used, labyrinth system can reduce groups into single files. The use of

sticks and rigid tubes must be avoided because they may cause deep bruising if

improperly used (Szent István University, 2009). Other devices less invasive such as

boards or paddles are always suggested in addition to a continuous training of stock

people about the correct handling of pigs during pre-slaughter operations and an

improvement of abattoirs structure.

Carcass and pork quality

Carcass and meat quality are essential criteria for food market and consumers of

pork. Genetics, production system, health, transport, handling, processing and

packaging, all contribute to the final result and among these factors, genetics can

determine between 30 and 60% of the total carcass and meat quality variation

(Chesnais, 1996; Andersen, 2000).

Skin bruises

The presence of carcass blemishes are indicative of poor welfare and, depending on

the severity, may cause carcass downgrading, up to 6% of its value (MLC, 1985), as

21

well as additional costs for reduction of the slaughter line speed to remove

damaged tissues and increased staff for carcass inspection. Further charges could be

attributed to loss of market opportunities as severely bruised carcass or traits are

rejected and can only be used for making lower value products (Faucitano, 2001).

The degree of damage on the skin of the carcass could be measured using different

scales, either providing a general score based on the carcass appearance (1 = none

to 5 = severe; MLC, 1985) or counting the bruises by anatomical location, usually

head/shoulder, middle/loin and ham (ITP, 1996; Barton-Gade et al., 1996a), defining

their shape and size to identify the source of infliction e.g., fighting, rough handling,

overcrowding and poor facilities (Faucitano, 2001, ITP, 1996).

Small bruises are normally linked to biting during fighting and are often placed in the

anterior (head/shoulder) (Weschenfelder et al. 2013). Conflicts among pigs can

results in blemished hams due to the combination of aggressivity and the

impossibility of avoiding an attack of a pig by another in high density pen (Faucitano,

2001, Geverink et al., 1996). Long and thin ones should be instead probably

recunducted to climbing during overcrowding situations (loading, unloading and

holding pen) and the moving along the slaughterhouse chutes, especially toward the

stunning (Weschenfelder et al. 2013, Faucitano, 2001) and are located on the back.

Rectangular and large dark brown marks are generally sticks-inflicted and

concentrated in the loin region, contrary of goads which tends not leave marks on

skin carcass, unless applied too widely (Dalla Costa et al., 2007, Faucitano, 2001).

The colour of the bruises too can give information on the time of infliction, with red

bruises being the recenter (Faucitano, 2001) than yellows ones.

22

pH

Skin bruises are then linked to post-mortem pH of meat. High bruise scores on the

carcass due to fighting in lairage or mounting behaviour results in higher ultimate

muscle pH due to the effects of physical activity on glycogen levels at slaughter

(Rabaste et al., 2007, Warriss 1996; Gispert et al. 2000). A positive correlation

between the presence of bruises score and pHu are also found by Romero et al.

2013, Vimisio et al. 2013 in pigs and Strappini et al., 2010 in cattle. When animals

are stressed, glycogen reserves can be depleted and hence a higher pH can be

expected (McVeigh & Tarrant, 1982). Multiple and prolonged stress caused by

improper handling, long transport, long fasting times and physical injuries can result

in depletion of muscle glycogen stores before slaughter, reducing acidification and

increasing ultima pH. McNally & Warriss (1996) reported that heavy bruised

carcasses presented pH values over 5.8 compared to light bruised carcasses,

suggesting a strong relationship between stress conditions, bruises and pH carcass

values (Strappini et al., 2010). Values of final pH (pHu) higher than 6 are indicative of

the DFD (dark, firm, dry) meat quality defect (Tarrant, 1989; Carragher and

Matthews, 1996; Gregory, 1998).

DFD and PSE defects

In DFD, the high pH results in relatively little denaturation of proteins, water is

strictly bound and little or no exudates is formed (Warriss, 2000). This is

because there is little or no shrinkage of the myofilament and the oxygen

penetration is reduced by the closed structure and results in a thin surface layer of

bright red oxygenated myoglobin (MbO2) and under the purple colour the

muscles (Adzitey and Nurul, 2011). This condition make appearing the meat darker,

with a dry texture and a less pronounced taste, characteristics less appreciated in

23

fresh cuts (Fisher, 2005). Furthermore, the high pH value, which is conducive to

bacterial growth, results in shorter shelf-life (Fischer, 2005, Holmer et al., 2009,

Faucitano et al., 2010).

Conversely the breakdown of glycogen just before slaughter lead to greater lactic

acid rate in the muscle inducing a fast pH decline (lower than 6 at 45 min post-

mortem) (Adzitey, 2011, Warriss and Brown, 1987), which if combined with high

muscle temperature, may result in the PSE meat defect (Gregory, 1998). This sudden

acidification provokes proteins denaturation and a consequently reduction in their

water holding capacity and releasing of exudates. The shrinkage of the

myofilament increases the light reflected from the meat and the haem pigments

selectively absorbed green light, reducing the normal red colour. This makes meat

paler (less red and more yellow), soft and exsudative (PSE) and with poor

palatability for reduced tenderness and juiciness after cooking (Adzitey and Nurul,

2011, Fischer, 2005). PSE is a meat defect caused by a combination of different

factors, such as stress-susceptible genes, rough handling shortly prior to slaughter

and poor carcass chilling (Grandin 2000a). Temperatures above levels of 37.0-39.6

(Hannon et al., 1990) result in increased PSE pork and a reduced shelf-life due to

increased chances of growth of microorganism (Lambooij, 2000), imposes negative

economic implications to the pork industry.

The literature describes that PSE defect could be also affected by the genotype. The

mutation known as the halothane gene (n) is responsible for genetic stress-prone

pigs (Gispert et al., 2000) and a linkage between the halotane gene (n) and an

increased incidence of PSE pork was shown by different studies (Fernandez et al.,

2002, Channon et al., 2000). The halothane gene has been demonstrated having an

additive effect with the other pre-slaughter stressors on PSE in meat (Channon et

al., 2000). The highest incidence of serious PSE carcasses was observed in pigs

slaughtered in a plant with the major n gene frequency. (Gispert et al., 2000).

24

The incidence of PSE pork could be reduced by controlling carcass chilling which is

able to influence the metabolism of muscle post mortem. A rapid fall of post-

slaughter temperatures reduces the speed of chemical and biochemical reactions

and the rate of pH decline. Nevertheless, , an extreme fast chilling may produce

tougher meat caused by cold shortening (Weschenfelder, 2013, Savell et al., 2005).

It is evident that pH values after slaughter greatly affect the final quality of meat

(Gajana 2013, Hambrecht et al., 2004, Gispert, 2000). The rate of pH decline could

adversely influence other pork characteristics as colour, water- holding capacity

(WHC), shelf-life and other technological properties i.e. cooking loss, tenderness,

juiciness, flavour (Gajana, 2012).

Colour

The colour impacts on meat sale because consumers use discoloration as a

parameter of freshness and healthiness of pork meat (Muchenje et al.2011, 2008,

Mancini and Hunt, 2005, Rosenvold and Andersen, 2003).

Meat colour is affected by elements such as feeding, age and physical activity of

animal. The concentration of pigments in muscle, mainly myoglobin, defines the

colour perception (Warner, 1994). Myoglobin store in the muscle changes between

species, breeds and single muscles and it can present three different chemical forms

(Gajana, 2012). Meat colour is usually detected by tricolorometric method where

colour coordinates for reflecting lightness (L*), redness (a*) and yellowness (b*), are

measured by a chromameter (Minolta) which evaluates the reflectance of the

myoglobin and haemoglobin after exposure to oxygenation (Weschenfelder, 2013,

Brewer et al., 2001).

25

Water-holding capacity

Water-holding capacity depends from muscle energy store at the moment of

slaughter and from the rate of pH decline (Van der Wal et al., 1999; Barbut et al.,

2008). It is defined as the ability of meat to retain its water which, in turn, influences

the juiciness. Short-term acute stress at the time of stunning induces a fast post-

mortem glycolysis and a storage of hydrogen ions and lactate meanwhile muscle

temperature is high yet. Low pH values at high temperatures lead to denaturation of

myosin and myofibrillar proteins (Warner et al., 1997; Joo et al., 1999) with a lower

water-binding capacity (Offer et al., 1989) (Muchenje, 2011). The quick driving and

coercion of pigs from lairage toward the stunning chute resulted in decreased water

holding capacity 24 h post-mortem (Muchenje, 2011, Van der Wal et al., 1999) and

consequently, in a higher drip loss. Practices as too long withdraw and excitement

and forced exercise may lower the water binding capability of proteins even when

the glycogen concentrations have been restored to normal during the post-mortem

period (Muchenje, 2011). Loss of water from meat has negative economic impact on

pig industry because it reduces the meat weight (Otto et al., 2007), water soluble

nutrients and negatively affects technological properties as toughness, pork flavour

and colour (Muchenje, 2011).

Drip loss

Drip loss is the inability of the muscle fibres to hold onto the natural juices of meat

with a water loss from the shrinkage of muscle proteins in the form of drip, even in

the absence of external forces (Weschenfelder, 2013, Fischer, 2007). This condition

changes the shape of pork through shrinkage and results in firmness and poor

juiciness (Muchenje, 2011). Drip loss measurement can be done by different

methodologies, such as the bag (Honikel, 1998), filter paper (Kauffman et al., 1986)

and EZ-Drip loss methods (Christensen, 2003) (Weschenfelder, 2013).

26

Some authors had found a significant correlation between genotype and this trait,

with higher drip loss in pigs with halothane-sensibility regardless of the type of

handling (Muchenje, 2011).

Cooking loss

Low water holding capacity (WHC) and low pH result also in high cooking loss

(Aaslyng et al., 2003; Muchenje, 2007). Cooking loss is the percent weight difference

between fresh and cooked meat samples (Muchenje, 2011, Honikel, 1998; Torley et

al., 2000; Moelich et al., 2003). During cooking chemical reactions, such as protein

denaturation and Maillard reaction shift physical meat characteristics involving also

a loss of water, several essential minerals and vitamins that influence its final

quality, acceptability (Chiavaro et al., 2009) and nutritional profile (Muchenje,

2011). Has been calculating that with cooking process, meat suffers losses from 20

to 40 % (Muchenje, 2011, Jonsall et al., 2001; Aaslyng et al., 2003; Muchenje, 2007).

The amount of cooking loss is highly dependent on cooking method, cooking

time/temperature and end-point temperature (Pearce et al., 2011, Aaslyng et al.,

2003), but is correlated to post-mortem pH too (Torley et al., 2000; Muchenje,

2007). Since pre-slaughter stress influences pork acidity, its water holding capacity

and structural muscle changes it may consequently affects also cooking loss

Muchenje, 2011).

Porcine meat from RN-gene pigs is associated with high cooking loss (>25%)

(Lundstrom et al., 1996; 1998; Jonsall et al., 2001) due to combination in meat of

higher glycogen content and a lower protein binding water during cooking

(Fernandez et al., 1991), Pugliese et al. (2005) confirmed that less cooking loss in

pigs which are kept under free-range conditions. High cooking loss is synonymous of

a less eating quality and implies financials losses for whole food industry (Muchenje,

2011, Aaslyng et al., 2003).

27

Flavour

Juncher et al. (2003) and O’Neill et al. (2003b) suggested that severe pre-slaughter

stress could enhance more drip and cooking loss as well as warm-over flavours

(WOFs) in PSE meat.

Among technological and sensorial meat quality parameters which can be

compromised by incorrect pre-stunning conditions is included the smell , that is a

fundamental component of product palatability, together juiciness and tenderness.

Juncher et al. (2003) found that following severe pre-slaughter stress pork can lead

changes in aroma, with development of warmed-over flavours (WOFs).

This flavour defect is due to a rapid phospholipids oxidations and protein

degradation that provoke the rancid-like flavour (Byrne et al., 2001, Byrne et al.,

1999), even if they could be masked up by the acidic flavours associated with PSE

meat (Muchenje, 2011, Gregory, 2007). Warmed-over flavour (WOF) is recognized

as a major quality problem by the meat industry in marketing of pre-cooked, ready-

to-heat and serve products (Byrne et al., 1999).

Tenderness

Pork tenderness could be indirectly affected by pre-slaughter handling, transport

quality, type of stunning, freezing and thawing (Gregory, 2007, Channon 2000) as

well as by breed, sex and age (Gajana, 2012). The cartilage and connective tissue

content of meat are further elements that impacting on meat tenderness

(Muchenje, 2011). Meat tenderness refers to the toughness or the ability of meat to

resist fragmentation when being chewed (Muchenje, 2011) and represent the most

important tasty characteristic. Warner–Bratzler shear force test is a well known

method to measure tenderness observed sensorial and to determine meat

acceptability (Gajana, 2012, Sanudo et al., 2003). Meat tenderness and texture are

important factors for consumers since they lay down the trend consumption and

28

purchase of meat. In order to achieve uniformity in tenderness and reduce

technologic defects in pork, it’s should improve and standardize ante-mortem

handling of pigs and post-mortem manipulation procedures of carcasses along the

chain (Gajana, 2012).

29

Aim

The aim of the thesis is clarify which are the factors that in pre-slaughter period can

play adversely on pigs welfare and meat quality.

In particular, this work focused the attention on transport distance and climatic

conditions, and on all these hazards which can negatively affect carcass and meat

quality parameters during unloading at the slaughter plant and the moving of

animals to the stunning cage.

Between carcass portions that could be damaged by inadequate pre-slaufhter

handling, raw ham has to be mostly considered, because it is destined to DPO chain

for dry-cured ham production, a very precious and high quality product of Italian

food industry.

In order to indentify the factors more involved in carcass damages, the Risk

Assessment (RA) was applied. It is a methodologyinstrument to identify these

factors considering a wide scenario and not only a single steps of pre.slaughter

procedures.

The RA approach used in the thesis is based on direct observation (mettere la

defiizione). It is totally non invasive and much indicates to work in a scenarios where

live animals are involved. If it is correctly applied, the RA could provide very useful

information to identify critical points in the whole pre-slaughter management,

solving carcass and meat quality damage problems.

The last goal of this thesis is remark the importance of the control and improvement

of pre-slaughter practices which are very important for the pig meat industry

economy.

30

CHAPTER 1

Risk characterization for carcass lesions during the pre-slaughter steps at

unloading and stunning of Italian heavy pigs

Agnese Arduini¹, Eduardo A. de Oliveira¹, Veronica Redaelli2, Fabio Luzi2, Vincenzo

Pace3, Leonardo N. Costa¹

¹ Dipartimento di Scienze e Tecnologie Agro-Alimentari, Università di Bologna, Italy.

2 Dipartimento di Scienze Veterinarie per la Salute, Produzione Animale e Sicurezza

Alimentare, Università di Milano, Italy.

3 Organizzazione Produttori Allevatori Suini (OPAS), Italy

Corresponding author: Agnese Arduini, DISTAL, Università di Bologna, viale Fanin,

46, Bologna (BO), Italy. Tel +39.051.2096513 – Fax +39.051.2096516. E-mail:

agnese.arduini@unibo.it

Abstract

Skin damage in pig carcasses are a welfare concern and can lead to rejection of

valuable cuts by DPO consortia and food industry. Carcass lesions can occur during

pre-slaughter period as a consequence of inadequate design facilities and rough

incorrect handling. The aim of this work was to provide, through the application of

RA (Risk Assessment), the identification of the main hazards which before slaughter

play the major role in the appearance of skin damage in the ham, loin and shoulder-

head of the pig carcass at the slaughterhouse. To this it was evaluated the

31

occurrence of backward, slips, balks, overlaps, falls, overcrowds, back up, pushing

and the use of rubber stick at unloading and during the moving to the stunning in

1680 Italian heavy pigs according to EFSA Guidelines on Risk Assessment. Frequency

of pigs behaviors was recorded for each deck unloaded and related to damage score

of the deck itself. For damage evaluation were considered the total carcass and the

ham, loin and shoulder-head singularly. For the total carcass at unloading, the use of

rubber stick showed the higher risk and frequency (1.97 and 18,75%). Backward,

slips, balks, overlaps, falls and overcrowd ranged between 0.11 and 0.02 with low

frequencies. Rubber stick reported the higher frequency (91.67%) and risk 46.29

toward the stunning and backward, back up, pushing, overcrowd, slipping and falling

showed lower values (between 0.01 and 0.77). Similar results were found for ham,

loin and shoulder-head where the stick was the parameter more correlated to

lesions than animals responses. It was conclude that, in the observed scenario, the

risk of total carcass lesions and for its single part was firstly related to the driving

tool both at unloading and toward the stunning, there is a very light correlation with

pigs behaviors.

Key words: brushes, pig handling, slaughter pig, electric prod, carcass

Introduction

The skin damage in pigs industry is a welfare and economic concern (Guàrdia et al.,

2009) and is responsible for great losses for the producers due to the downgrading

of the carcass and rejection of valuable cuts like ham and loin by highly

sophisticated markets, the reduction of the production of dry cured hams (Candek-

Potokar and Skrlep, 2012), which need to be completely unblemished and of the

speed of dressing line to remove blemished tissues (Faucitano, 2001). Furthermore

the carcass lesions are associated with poor meat quality: pigs with high score of

skin damage tends to have higher levels of cortisol, creatine phosphokinase and

32

lactate (Warriss et al., 1998) and the stressful handling associated with carcass

lesions can predispose the occurrence of DFD or PSE meat (Rosenvold and

Andersen, 2003).

Factors as stress susceptibility and aggressiveness can influence incidence of carcass

bruises (Bolhuis et al. 2005) as well as loading and unloading practices and their

duration, time of transportation and stocking density (Nanni Costa et al. 2002, Nanni

Costa et al. 1999, Barton-Gade & Christensen 1998, Franqueza et al. 1998). Increase

of skin damages carcasses was demonstrated also for time of lairage and fasting

before slaughter (Guardià et al. 2009), mixing unfamiliar pigs (Brown et al. 1999) and

use of moving device such as electric prod (Rabaste et al. 2007, Guise and Penny,

1998). However, in general, the main causes are related to aggression, harsh

handling and poor design facilities (Faucitano, 2001), but it is not clear which are the

main points where may occur these injuries and for which specific event.

Some studies tried to identify the critical steps during ante mortem period for skin

damage on pigs (Romero et al. 2013 and Chevillon et al. 1997) and on culled cows

(Strappini et al. 2013) with different approaches. However, there is not a

standardized procedure to evaluate the real causes of carcass damage.

Risk Assessment (RA) is a systematic process consisting into identify and

characterize potential hazards (e.g. to animal welfare or food safety), to estimate

the probability and the magnitude of adverse effects resulting from exposure to

those hazards and to determine the resulting risk (Dalla Villa et al. 2009).

The risk assessment approach includes four steps: hazard identification (factors that

have the potential to create adverse effect or consequences), hazard

characterization (quantitative estimate of the adverse effect), exposure assessment

(qualitative or quantitative evaluation of the level, duration, and variability of

exposure to the identified factors) and risk characterization (qualitative or

33

quantitative estimation of the probability of occurrence and magnitude of known or

potential adverse effects) (EFSA, 2009; Algers et al., 2009). In pig production, the RA

has been carried out to evaluate meat quality (Guàrdia et al., 2005; Guàrdia et al.,

2004), foodborne zoonoses (Fosse et al., 2008) and animal diseases (Berends et al.,

1996), but never during pre-slaughter period in function of carcass lesions.

In this context, the aim of this work was to provide, through the application of risk

assessment, according the European Food Safety Authority (EFSA, 2012), new

information on the detrimental effects due to improper handling during all

operations involved in the slaughter of heavy pig that can induce lesions in the

whole carcass and single traits as ham, loin and shoulder-head.

Material and Methods

Risk Assessment Application

The study was conducted following the EFSA methodology on Risk Assessment for

Animal Welfare (EFSA, 2012).

The scenario observed was a slaughterhouse of medium dimensions, speed chain of

280 pigs/hour and specialized in the slaughter of Italian heavy pigs for the

production of Parma dry-cured ham. The plant was located in Lombardy and all

supply farms are from Northern Italy.

Potential causes of lesions (hazards) were identified in the target population made

of 12 deliveries of Italian heavy pigs (a total of 1680 animals, 140 for each journey)

from the same commercial farm over the winter, spring and summer of 2014. At the

farm, outside the boxes, the pigs were conveyed in an external passageway of

concrete material, long 60 m and linked to a mobile ramp (length m 6.0, width m

0.7, solid side walls of m 1.0 and adjustable height), using to load pigs into the

trucks. The pigs were loaded in the beginning of the morning after 10 hours of

fasting.

34

The transports were carried out with double trailer lorries with three hydraulic

decks each one. All compartments in the trailers were stocked according to the

Standard Regulations Operating Procedure (Reg. 1/2005 CE, 2005) with a density of

about 0.68 m²/pig. The pigs were transported through rural and secondary roads

and the journey time was about 30 minutes at an average speed of 60 km/h. At the

slaughterhouse, the unloading was carried out by a fixed bay adjustable for the

height and performed by the driver and the manager of the slaughter lairages, in

around 20 minutes. After unloading, pigs were immediately moved to the stunning

chute or into resting pens for a maximum time of 30 minutes.

When driven to the stunning, pigs were handled with rubber sticks by a slaughter

operator until the single chute leading the stunning cage, where pigs were driven

only by electric prod. Pigs were electrical stunned (head only-1,3A and 170V) and

exanguinated horizontally for 3 minutes. After sticking, pigs were hanged by the left

leg for 10 minutes before being immersed in a scalding tank for dehairing at 62°C for

5 minutes and the remained hair was manually removed after. The cleaned

carcasses were evaluated for skin damages, assessed subjectively by the same

trained operator, on the ham, loin and shoulder-head as Velarde et al. (2001) and

Chevillon et al. (1997) by a four point scale (1=none to 4=extreme) according to the

Danish Meat Reasearch Institute-scale (Barton Gade et al., 1996). Carcasses were

then eviscerated, split, hot-boned and sectioned in different marketing pieces,

before kept in chilling rooms (2–4 °C) for 24 hours.

Hazard measurement

The target of hazards for skin bruises was obtained merging events directly

observed at the slaughterhouse and those reported in literature about lesions on

the carcass. According to Dokmanovic et al. (2014) and Aiassa (2010) for pigs and

Strappini et al. (2013) for bovine, operators handling and some pigs behaviors were

35

observed and recorded together at unloading and during moving toward the

entrance of stunning chute (Table 01 and Table 02).

Table 1. Descriptions of behaviors observed.

Behavior Description Slips Pig's leg touching the ground Falls Pig’s body touching the ground Back up Pig moves rearward, opposite the direction of intended motion Backwards Pig makes a 180° turn and moves in the opposite direction Overlaps Pig mount another pig, with its front legs on the back of the other

pig Overcrowd More pigs huddle and create a group Balks Pig refuse to walk or stops for greater than 2s Pushes Pig press the head against another/other pigs in front of him Rubber stick Handler uses rubber stick to encourage pigs to move

Data were recorded and evaluated considering the total number of events per deck

of the truck, which was the experimental unit.

Data Analysis

The risk was estimated by multiplying the probability of occurrence (PO) for the

Magnitude, according to EFSA (2012):

Risk = Probability of Occurrence x Magnitude

The probability of occurrence was considered multiplying the Distribution of

Exposure by Distribution of Likelihood.

Probability of Occurrence = Distribution of exposure x Distribution of likelihood

For the Distribution of Exposure was used the frequency recorded of each event on

total number of pigs observed, expressed as percentage, as used by Fosse et al.

36

(2008). As the Distribution of Likelihood was considered the ratio between the total

number of each event and the sum of mean number of pigs with lesions at carcass,

ham, loin and shoulder-head respectively for each deck.

Distribution of Exposure = Frequency of each event

Frequency of each event = Total number of each event/Total number pigs

Distribution of Likelihood = Total of each event/Sum of bruising score of total carcass

The Magnitude of the adverse effect, i.e. the potential adverse effect at the

individual level, given that the animal is exposed to the hazard and experiences that

adverse effect, was calculated multiplying Severity x Duration (EFSA, 2012; EFSA

2009), adjusting the levels in order to give weighting to the scores. As performed by

Dalla Villa et al. (2009) that estimated Severity levels subjectively, a scale from 1 to

4 was chosen for assessing severity, based on the effect of the parameters observed

on carcass lesions in the scenario.

Magnitude = Severity / 4

In the present study the duration was fixed to 1 for each hazard, because the direct

observation of each events lasted always less or equal to 1 minute for the speed of

the chain.

Table 2. Formulas used to estimate the final Risk (Dalla Villa et al. 2008)

Magnitudo = Severity/4 *Duration Frequency = Total of each event/Total number of pigs Distr. Of Likelihood = Total of each event/Total of mean bruised pigs Distr. Of Exposure = Frequency Prob. Of Occ. = Distr. Of Likelihood * Distr. Of Exposure Risk = Magnitudo * Prob. Of Occurrence

37

A cluster analysis (3 groups) and a dendrogram graphic were performed with

unloading and stunning variables correlation’s with total carcass lesions in order to

better understand the type of connection among the parameters observed (hazards)

and the total score of bruises.

Results and Discussion

All factors potentially causes of lesions in carcass were analyzed into two groups,

corresponding to the moment of observation, unloading and going to the stunning

raceway for total carcass, ham, loin and shoulder-head respectively.

Carcass lesions

Table. 4. Classification of parameter for Risk of lesions on the total carcass

Parameters Severity Magnitudo Frequency % Distr. Likelihood Prob. of Occ. Risk

Unloading

rubber stick 2 0,5 18,75 0,21 3,94 1,97 backwards 1 0,25 6,49 0,07 0,45 0,11 slips 1 0,25 3,57 0,04 0,14 0,04 falls 2 0,5 1,73 0,02 0,03 0,02 balks 1 0,25 2,8 0,03 0,08 0,02 overlaps 2 0,5 1,96 0,02 0,04 0,02 overcrowds 1 0,25 2,2 0,02 0,04 0,02

Toward Stunning rubber stick 2 0,5 91,67 1,01 92,59 46,29 backwards 2 0,5 11,96 0,13 1,55 0,77 back ups 2 0,5 6,9 0,08 0,55 0,28 pushes 2 0,5 1,96 0,02 0,04 0,02 overcrowds 2 0,5 1,73 0,02 0,03 0,02 slips 2 0,5 1,01 0,01 0,01 0,01

Distr=Distrubution; Prob=Probability.

For the total lesions of carcass at the unloading, the rubber stick was the only tool

used by the handlers and the event with major frequency (18.75%), probably of

occurrence (PO) of the adverse effect (3.94) and the higher risk (1.97) associated to

38

lesions. The result agree with Chevillon et al. (1997) and Geverink et al. (1996), who

reported a relationship between the use of stick and carcass lesions. The use of this

device is just a routine and a reaction of handlers impatience for hesitation of pigs

caused by novel sounds and environments connected to all transport’s operations

induce an high use of stick for moving on animals.

The transfer from the familiar pen to the novelty of the truck inner and the abattoir

area, joined to the high physical activity induced by the costraint to go trought

sloped ramps and along alleys by stockpersons, may cause fear in pigs, evoking

reluctance to move on or try to reverse (Dalmau et al. 2009, Faucitano 2001).

Moreover is known that is practice of transport workers and abattoir employees

make excessive use of sticks and/or goads to force and move pigs quickly the last

metres prior to the stunning to mantain the rhythm of slaughter. (Dalmau et al.

2009, Faucitano 2001).

Pigs behaviors as backwards, slips and balks showed frequencies between 6.49 and

2.80 %, a PO ranged 0.45 and 0.08 and a negligible risk values, probably due to an

indirect connection with skin bruises. Overlaps, falls and overcrowd showed the

lowest frequencies, PO and risk values not relevant for lesions, unlike found by

Weshenfelder et al. (2012) and Torrey et al. (2008), who justified greater skin

damage by the great frequency of overlaps, slips and falls in this step. The PO of

adverse effect was too low because these actions non strictly induce bruises.

Going to the stunning chute, the rubber stick was again the parameter with the

highest frequency (91.67 %) and highest risk to cause lesions in carcass (46.29). It is

well known that this tool, as well as electric prod, increase incidence of blemished

carcasses (Rabaste et al. 2007, Guise and Penny 1989, Faucitano et al. 1998) and its

overuse immediately before slaughter could decrease animal welfare and the

glycogen stores in the muscles with a worsen quality of meat due to the occurrence

39

of PSE (pale, soft, exudative) (Rabaste et al., 2007). This data reflect the trend of

abattoir employees that, for impatience and the need to respect the slaughter

speed, make an improper use of sticks and goads, especially near the stunning race

(Faucitano, 2001) inducing panic in pigs and potential deep bruising in carcass

(Szoucs et al. 2008).

The frequency of backwards along the alleys until stunning raceway gate was higher

than at unloading (11.96 and 6.49, respectively), probably due to the fear of animals

for novel, increased noise combined with the presence of employees with tools

(stick) in a closer space as the restrainer pen: is just documented that, in reduced

area and in presence of stress factors, pigs try to escape, changing the direction

(Rabaste et al, 2007). The PO for backwards was calculated equal to 1.55 and the

estimated risk of lesions 0.77. This behavior, not directly correlated to skin damage,

could induce handlers that the use stick or rigid tube often too much and harshly to

drive and separate pigs to the final single chute (Rabaste et al., 2007) maintaining

constant the speed chain (Faucitano, 2001), but may causing deep bruises (Szucs et

al. 2008).

Back up, observed only inside the restraining pen, had a frequency of 6.90 %, due to

the very limited area for movement where can entry only 5-6 pigs. Moreover, the

high use of the stick by the handler may had reduced this behavior of animals, that

scared, usually went quickly on and entered inside the single chute. As consequence,

the PO and the connected risk on carcass lesions were low (0.55 and 0.28).

Pushes, overcrowds, slips and falls showed the lowest frequencies probably

explained by the fact that, in this scenario, the animals were quite calm. The risk of

lesions was almost nonexistent, maybe because the events are more linked to

animal welfare issue than directly to lesions one.

40

Lesions on ham, loin and shoulder-head

For the ham, at unloading the rubber stick remained the main hazard of lesions with

the highest PO (3.94) and risk (1.97), reporting the same values than for total

carcass. Pigs behaviors also reported a similar trend of the whole carcass with the

backwards that was the second events connected to lesions (PO=0.48, risk=0.12)

and the other behaviors with lower values, ranged between 0.15 and 0.04 for the

PO and 0.04 and 0.02 for the risk, without any apparent connection with damage to

this trait.

A similar situation occurred along the passageways toward the stunning, with the

highest PO and risk found by the stick: 94.42 and 47.21, respectively. The behavioral

parameters showed a low PO and risk values minimal and not relevant for lesions to

the ham. However, it’s true that during fighting’s , that could happened in lairage of

the plant, pigs removed are more susceptible to lesions in the rear of the body

(Turner et al. 2006) and could be responsible of the lesions found in the hams.

For the loin area, at the unloading the rubber sick resulted the main hazard of skin

lesions with PO and risk equal to 3.67 and 1.83 respectively. Backwards showed

about the same numbers of the total carcass and the ham (PO=0.44 and risk=0.11),

slips, balks, falls, overcrowds and overlaps ranged from 0.13 and 0.04 of PO and 0.03

and 0.02 of risk. For the moving to the stunning, the rubber stick recorded was the

highest value (87.68) of PO as well as the risk (43.84), , followed by backward (1.49

and 0.75) back up, pushes, overcrowds and slips that had PO values among 0.50 and

0.01 and risk ranged from 0.25 to 0.01. These results not agree with Faucitano

(2001), who reported that typical behavioral responses of pigs before slaughter

make hard to handle them and create lacerations in skin, loin too.

At unloading and toward the stunning, also for shoulder-head the use of rubber stick

was the major event linked to lesions (PO=87.68 and Risk=47.67). This data is

41

probably due to the fact that the frequency of this parameter, as the other, was

recorded when observed independently if it caused effectively lesions and in which

part of the carcass. In fact the use of driving tools in this part of pigs’ body was never

seen in this scenario, also because its use toward this part could induce fear in pigs,

who scared, wouldn’t go on the pathway, but rather try to escape. Although the

results, its possibly that the lesions recorded in this area are due to pre-slaughter

fighting (Faucitano, 2001) or pigs’ agitation, that jamming in the close passageways,

injured themselves with contact of the concrete walls of alleys.

For animals behavior, backwards was in both two steps the factor more connected

with lesions to this area, with highest values of PO and Risk, 0.45 and 0.11 at

unloading, 1.67 and 0.84 to the stunning respectively; lesions could be caused by

impacts of animals with the facilities during the backwards. Back up reported PO

and Risk values highest of ham and loin (0.58 and 0.29) maybe explained with the

fact that, once forced to move, pigs tended to overcrowd, jamming each other and

beating with structures, especially against the gate of stunning chute. The same

reason could explain the values of PO and risk, even if low, of pushes, overcrowds

and slips (PO=0.05, 0.04 and 0.01 and Risk=0.02, 0.02 and 0.01, respectively) usually

responses to anxiety and attempt to escape provoked by the need of operators to

dissolve the group and reduce it to a single file (Faucitano, 2001).

42

Table. 5. Classification of parameter for Risk of lesions on ham, loin and shoulder-head at unloading.

Parameters Severity Magnitudo Frequency% Distribution of likelihood Probably of Occurrence Risk

ham, loin and shoulder-head ham loin shoulder-head ham loin shoulder-head ham loin shoulder-head

rubber stick 2 0,5 18,75 0,21 0,20 0,21 3,94 3,67 3,94 1,97 1,83 1,97 backwards 1 0,25 6,49 0,07 0,07 0,07 0,48 0,44 0,45 0,12 0,11 0,11 slips 1 0,25 3,57 0,04 0,04 0,04 0,15 0,13 0,16 0,04 0,03 0,04 balks 1 0,25 2,80 0,03 0,02 0,03 0,09 0,08 0,10 0,02 0,02 0,02 falls 2 0,5 1,73 0,02 0,03 0,02 0,03 0,03 0,04 0,02 0,02 0,02 overcrowds 1 0,25 2,20 0,02 0,02 0,02 0,04 0,05 0,04 0,01 0,01 0,02 overlaps 2 0,5 1,96 0,02 0,02 0,02 0,04 0,04 0,05 0,02 0,02 0,01

Table.6. Classification of parameters for Risk of lesions for ham, loin and shoulder-head toward the stunning.

Parameters Severity Magnitudo Frequency% Distribution of likelihood Probably of Occurrence Risk

ham,loin and shoulder-head ham loin shoulder-head ham loin shoulder-head ham loin shoulder-head

rubber stick 2 0,5 91,67 1,03 0,96 1,04 94,42 87,68 95,34 47,21 43,84 47,67

backwards 2 0,5 11,96 0,13 0,12 0,14 1,55 1,49 1,67 0,77 0,75 0,84

back ups 2 0,5 6,90 0,08 0,07 0,08 0,55 0,50 0,58 0,27 0,25 0,29

pushes 2 0,5 1,96 0,02 0,02 0,02 0,04 0,04 0,05 0,22 0,02 0,02

overcrowds 2 0,5 1,73 0,02 0,02 0,02 0,03 0,03 0,04 0,02 0,02 0,02

slips 2 0,5 1,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01

43

Considering the risk characterization a sort of classification of the potential hazards,

the cluster analysis gave a clearer representation of the linkage of the variables with

lesions in general, as Arandom et al. (2012) who evaluated the relation among stress

behaviors of pigs and blood parameters and transport time.

At unloading the use of stick is clearly connected to some pigs behaviors, as slips,

falls, overcrowds and balks that are probably induced to it. Otherwise backwards

and overlaps results in a single cluster each one because may be explained only by

the fear of pigs of the new external environment (Figure 1).

Figure 1. Dendogram of unloading hazards and total carcass lesions

The movement toward the stunning raceway (Figure 2) showed that all pigs

behaviors, excepted the backwards, were connected together and, probably, one

the cause of the other, due to the well-known stress and anxiety typical of this

phase. Backwards not clustered with other pigs’ parameters with which has no

44

relation, but is linked to lesions perhaps the hits of pigs during backwards versus

different surfaces of the plant, in the attempt to avoid to go on.

The use of rubber stick results in a single cluster indicating that operators’ activity

not depended from animals’ responses, but tend to be routine behavior even if

often excessively and aggressively acted that could be the direct cause of skin

lesions in pigs body.

Figure 2. Dendogram of hazards toward the stunning and total carcass lesions

Conclusions

In the observed scenario, the risk of carcass lesions was mainly related to the use of

rubber stick. The operator handling confirmed as an important hazard for the

45

presence of bruises of the carcass during pre-slaughter phases, especially near the

stunning where the animals management result more difficult and severe.

These results suggest that improving staff’s training and providing better conditions

during the driving of pigs at slaughterhouse could be an effective device to reduce

the incidence of skin lesions and the consequent economic losses, as well improve

animal welfare. It’s well known that flags and paddle are more recommended tools

for driving pigs, at contrary of sticks, goads and prod. However, all steps of pigs

handling prior to slaughter should be taken into account, including loading at farm

and transport, because lesions may be caused also in these critical phases.

Further studies should be done to identify the real causes of lesions and the

moment of their infliction to reduce carcass damages and increase pigs welfare.

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51

CHAPTER 2

Effect of Transport Distance and Season on Some Defects of Fresh Hams

Destined for DPO Production

Agnese Arduini 1,*, Veronica Redaelli 1, Fabio Luzi 2, Stefania Dall’Olio 1, Vincenzo

Pace 3 and Leonardo Nanni Costa 1

1 Department of Agriculture and Food Technology, School of Agriculture and

Veterinary Medicine, University of Bologna, Via Fanin 50, 40127 Bologna, Italy; E-

Mails: veronica.redaelli@unibo.it (V.R.); stefania.dallolio@unibo.it (S.D.);

leonardo.nannicosta@unibo.it (L.N.C.) 2 Department of Health, Animal Science and Food Safety, Faculty of Veterinary

Science,

University of Milan, Via Celoria 10, 20133 Milan, Italy; E-Mail: fabio.luzi@unimi.it 3 OPAS, Pig Farmer Association, Strada Ghisiolo 57, 46030 San Giorgio, Mantua,

Italy;

E-Mail: vincenzo.opas@coopgsp.it

* Author to whom correspondence should be addressed; E-Mail:

agnese.arduini@unibo.it;

Tel.: +39-051-2096-594; Fax: +39-051-2096-596.

Received: 7 June 2014; in revised form: 15 August 2014 / Accepted: 25 August 2014 /

Published:

Simple Summary: Transport to the slaughterhouse is a stressful event for pigs.

Travel duration and conditions can negatively affect animal welfare and carcass

quality. Some defects in fresh hams are strictly connected to pre-slaughter

transportation. Journeys with short (<37 km) and long (>170 km) distances may

increase damage in fresh hams and decrease Denomination Protected Origin

52

(DPO) Parma dry-cured ham production.

Abstract: Pre-slaughter handling is related to defects in fresh hams that result

in exclusion from the DPO (Denomination Protected Origin) Parma chain,

including hematomas, lacerations, microhemorrhages and veining. To

determine the effects of transport conditions on hams, we collected data on

defects in 901,990 trimmed fresh hams from heavy pigs provided by 3,650

batches from slaughterhouse during 2012 and 2013. For all batches, transport

distance (1–276 km) season and year of delivery were considered.

A decrease of all defect occurrences was observed for increasing distance up to

170 km (P < 0.05). Above 170 km, however, all defects frequencies increased (P

< 0.05). Season showed an effect on the incidence of defects, with an

increasing of hematomas and lacerations in winter and autumn respectively (P

< 0.05) and the highest percentage of veining and hemorrhages in spring (P <

0.05). Summer had the lowest incidence of defects on fresh hams. We

concluded that the incidence of the examined defects and the subsequent

rejection for DPO Parma ham production is lower in fresh hams transported

38–170 km during the summer.

Keywords: pigs; transport; distance; season; DPO Parma ham; pre-slaughter

handling; defect

1. Introduction

Pre-slaughter is known to be a critical period for pigs [1,2], because several factors,

i.e., inadequate transport conditions, inappropriate handling [3] and length of the

journey [4], can seriously affecting animals welfare both physically and

psychologically. Additionally, carcass quality can be adversely affected by

inadequate pre-slaughter conditions, such as rough practices of loading and

unloading and inappropriate driving style of trucks and pigs that may lead to skin

53

wounds, hemorrhages and hematomas [2]. Increased incidence of blood-splashed

and skin damage was found in pigs handled roughly or driven by electric prods

during pre-slaughter [5]. These injuries represent a significant economic loss,

especially if they are located on the most valuable cuts such as hams, loins and

shoulders. According to Von Borrell (2005) [6], transport is considered to be a major

stressor might have deleterious effects on product quality. Previous research

indicates that pig welfare is affected by both long and short journeys [7]. Mota-Rojas

et al. [8] found that the percentage of bruised carcasses increases with journey

duration, while Barton Gade and Christensen [9] reported an increased risk of skin

damage during short transports (2–3 h), and Gispert et al. [10] found higher skin

damage scores in short transports (<2 h) vs. long transports (>2 h).

The effect of the season on pig welfare and carcass quality have been also reported

[11,12]. The frequency of skin blemishes, hematomas and blood splashing was

shown to increase in winter, probably caused by slips and falls due to slippery icy

floor [13–15].

Raw hams from heavy pigs (160–170 kg live weight) reared in Northern Italy are

primarily processed to DPO (Denomination Protected Origin) dry-cured hams under

the control of several consortia, of which Parma Consortium is largest, branding

about 10 million dry-cured hams per year (PQI 2014, PQI 2013, Prosciutto di

Parma.it) [16,17]. The Parma Quality Institute (PQI) [16] inspects fresh hams

destined for the DPO dry-curing process for the presence of an inappropriate

physical characteristics like lean-fat ratio or physical injuries, such as hematomas,

microhemorrhages, muscle lacerations and subcutaneous veining caused by pre-

slaughter handling; these lesions lead to exclusion of the cuts from the DPO Parma

chain. A study of more than 100,000 fresh hams monitored by PQI in 2013 found

that 4.3 % of these were rejected for DPO dry-cured ham production due to

hematomas, microhemorrhages, muscle lacerations and subcutaneous veining.

54

Extended to the annual production of fresh hams destined to DPO Parma dry-cured

ham equal to 16 million, the total number of fresh hams rejected for pre-slaughter

defects could reach 689,000 with an estimated economic loss around 7.6 million

euro per year for the whole Parma Consortium and Italian Food Industry.

The aim of this study was to evaluate the effects of transport distance, in connection to

the slaughter season, on the incidence of defects observed on fresh hams destined for

processing to DPO dry-cured ham.

2. Experimental Section

2.1. Data Sampling

This study was carried out in 2012 and 2013 on data collected from 901,990

trimmed fresh hams of heavy pigs provided in 3,650 batches supplied by 411

farmers and slaughtered over 396 days in a single plant located in Northern Italy.

Each batch contained 7–151 pigs with an average of 124 animals per batch. All pigs

were 9 months old with live weights of kg 171.1 ± 6.1, as required by the Parma dry-

cured ham Consortium [18].

2.2. Pre-Slaughter Conditions and Abattoir

Transportation distances from farm to slaughterhouse ranged from 1 to 276 km and

average shipping speed based on motorway proximity and road conditions was

estimated at 60 km/h. The deliveries were carried out using double trailer lorries

with three hydraulic deck. The lorries had natural and mechanical ventilation

system, with fans placed on the left side of the trucks. Stocking density during

transport was estimated ranging 141–251 kg/m2 and the journey duration ranged

0.25–5 h. The loading was done using the mobile ramp commonly present at the

piggery (length m 6.0, width m 0.7, with solid side walls of m. 1.0 and adjustable

height), while unloading at the slaughterhouse was carried out by a platform (length

55

m 9.3, width m 2.7, solid side walls m 1.0) with adjustable height at the level of the

lower deck. The vehicle was always unloaded before the rear trailer. Lairage time

ranged between 30 min to 1.5 h. During this period, pigs were not mixed with

unfamiliar animals. Averages and ranges of seasonal temperature and relative

humidity during 2012 and 2013 in the area where pigs were transported are shown

in Table 1.

Table 1. Temperature (°C) and Relative Humidity (%) in the slaughterhouse area

during 2012 and 2013.

Temperature (°C) Relative Humidity (%)

2012 2013 2012 2013

Average Range Average Range Average Range Average Range

Winter 3 −2 9 4 0 8 81 58 100 85 69 100

Spring 17 11 23 16 10 21 70 44 100 73 50 100

Summer 24 17 32 23 16 30 66 39 100 70 42 100

Autumn 10 6 15 10 7 15 83 65 100 86 68 100

Pigs were driven with plastic sticks or rubber boards to resting pens, where they

remained 0.5–4.0 h, with only a few batches resting overnight. After resting, pigs

were showered and driven through a single chute to the stunning cage. Stunning

was accomplished by electrical tongs (head only; 170 V, 1.3 A). Carcasses were then

exsanguinated horizontally for 3 min and hanged vertically for 10 min before being

immersed into the scalding tank. Carcasses were hot-boned and kept in the cooler

(2–4 °C) overnight. The next day, hams were trimmed to the commercial shape,

classified for damages and selected according to market criteria.

56

2.3. Defects Observed

Data on defects such as hematomas, lacerations, microhemorrhages and superficial

veining related to pre-slaughter practices were recorded on trimmed fresh hams.

Hematomas are lumps formed by blood clots beneath the skin, typically associated

with ruptured blood vessels, that can be produced by impacts against either the

handling facilities or improperly used handling prods [3]. Lacerations are similar to

dark hematomas distributed in the medial side of the ham and are caused by muscle

tears, which may be related to slipping on wet floors during driving.

Microhemorrhages are characterized by pinpoint bleeding in the muscles due to

capillary rupture and are generally attributed to rough handling, electrical stunning

and vertical exsanguination [19–22]. Superficial veining is a subcutaneous venous

lattice affecting the medial, or sometimes the entire, surface of the thigh. This ham

defect appears several hours after slaughter, is particularly noticeable during the

dressing process and is still evident at the end of the processing and seasoning [23].

The incidence and severity of the veining defect was found to be associated with the

prolongation of pre-slaughter procedures (loading, transport and lairage time), with

the use of CO2 stunning methods and with the increase of time between the

separation of ham from the hot carcass and chilling from 15 min to 60 min [23];

however, the causes remain largely unexplained [19].

Trimmed hams were classified on the basis of the presence of defects, according to

PQI photographic standards, by two trained slaughterhouse operators and recorded

in an electronic database according to batch and producer identity. Defects were

calculated as percentage per batch.

2.4. Statistical Analysis

Batches were classified by distance between supplier and slaughterhouse using the

FASTclust procedure of Statistical Analysis System (SAS 9.3 Cary, NC, USA, 2009).

57

Data were assigned to four clusters based on the smallest Euclidean distance from

the initial seed in the cluster. The number of batches assigned to each cluster and

the respective mean distances, standard deviations (SD) and minimum (min) and

maximum (max) distances are reported in Table 2. Slaughter days were grouped into

seasons and were monitored over two years to evaluate defect incidence change

from year to year. The distribution of batches and pigs between season and year is

reported in Table 3. Defect incidences approximated a Poisson distribution and were

log transformed by the GLIMMIX procedure (SAS 9.3 Cary, NC, USA) prior to

statistical analysis. The model included cluster, season and year and their respective

interactions as fixed effects, and farm within day of slaughter as random effects. The

ILINK option was used to back-transform least squares means, and differences

between least squares means were evaluated by Tukey-Kramer’s test (P < 0.05).

Table 2. Distance and transport conditions for batches and pigs based on cluster.

Cluster (Distance) 1 (11–37 km) 2 (38–86 km) 3 (89–170 km) 4 (199–276 km)

No. of batches 1,573 990 347 740

No. of pigs 195,596 119,213 42,554 93,632

Average No. pigs/batch 124 120 123 127

Table 2. Cont. Cluster (Distance) 1 (11–37 km) 2 (38–86 km) 3 (89–170 km) 4 (199–276 km)

Distance:

-average (km) 21 50 121 237

-standard deviation (km) 10 9 18 12

-min (km) 1 38 89 199

-max (km) 37 86 170 276

Transport condition:

-type of vehicle:

58

single vehicle 298 246 74 112

double trailer 1,275 744 273 628

-estimated duration (h) 0.5 1–1.5 2–3 3–5

-average stocking density (kg/m²) 213 206 207 218

Table 3. Distribution of batches and pigs based on season and year.

Cluster (Distance) 1 (11–37 km) 2 (38–86 km) 3 (89–170 km) 4 (199–276 km)

Season

Winter

No. of batches 384 200 103 172

No. of pigs 47,765 24,581 12,881 21,759

Spring

No. of batches 401 266 87 190

No. of pigs 49,563 31,662 10,842 24,181

Summer

No. of batches 415 256 71 176

No. of pigs 50,496 30,659 8,458 22,608

Autumn

No. of batches 373 268 86 202

No. of pigs 47,772 32,311 10,373 25,048

Year

2012

No. of batches 780 433 202 373

No. of pigs 96,406 52,193 26,148 46,808

2013

No. of batches 793 557 145 367

No. of pigs 99,190 67,020 16,406 46,824

59

3. Results and Discussion

In 2012, the incidence of hematomas, lacerations and microhemorrhages observed

in trimmed hams were 4.3%, 1.7% and 1.5%, respectively. In 2013, hematomas

increased to 6.2%, while lacerations and microhemorrhages showed very similar

incidences equal to 1.7% and 1.6%, respectively. During the two years, PQI, detected

in the whole Parma chain (around 4200 farms and 130 slaughter plants) about twice

as much the incidence of these defects [16].

The results of variance analysis and the least-squares means of defects frequency by

sources of variation are reported in Table 3. Distance, season and year showed to

have a significant effect on the incidence of defects under study. Significant

interactions between distance × season and between distance x year were found for

hemorrhages, and for hemorrhages and veining, respectively. Moreover, there was

a significant season x year interaction for hemorrhages, veining and laceration.

Incidence of defects decreased with increasing transport distance from Cluster 1

(11–37 km) to Cluster 3 (89–170 km) (P < 0.05). The higher incidence of defects

associated with short transport distances may be due to the lack of time to lie down

and recovery from loading stress. Also, a reduction of standing pigs and increasing

journey duration could explain this result. Lambooij (2007) showed that the

percentages of slaughter pigs standing during transport decreased with journey

time, which leads to a reduction in risk of slips, falls and overlaps [2]. Additionally,

these results could be the consequence of truck drivers working hurriedly to

accomplish all planned transports for a given day, as well as the effect of the lower

quality of rural roads, which represent the major part of the route in short journeys.

Nevertheless, extending of transport distance above 170 km (Cluster 4) results in an

increased incidence of defects, which reaches and exceeds the frequency recorded

for Cluster 1. There were no differences in hematomas and veining between clusters

4 and 1, while lacerations and hemorrhages incidences were significantly higher (P <

60

0.05) in Cluster 4. Thus, the incidence of defects reported herein was not

proportional to the increase of transport distance. Mota-Rojas et al. [8] observed an

increase in pig carcass bruising prolonging the journey from 8 to 23 h. Gallo et al.

[24] identified similar trends in slaughter beef transported for 36 h. The incidence of

defects on fresh hams was also influenced by season of transport (Table 4). The

percentage of hematomas was greatest (P < 0.05) in winter, decreasing in spring and

summer (P < 0.05) and reaching the lowest incidence in autumn (P < 0.05). There are

no differences between winter, spring and summer for lacerations, while the

incidence of this defect was greater (P < 0.05) in autumn. Gosàlvez et al. [15]

reported a higher incidence of bruised carcasses in pigs transported in winter and in

autumn. Scheeren et al. [13] hypothesize that the greater proportion of skin damage

in winter is linked to the tendency of pigs to stand during transportation to avoid

making contact with the cold floor and walls of the trailer. Dalla Costa et al. [25]

found that pigs were more difficult to handle in winter and need more coercion,

which led to more handling-induced bruises. The higher incidence of lacerations

observed in autumn could be related to the increase of live weight in this season,

due to a restarting of feed intake for the decrease of temperature with respect to

the summer. The average live weights recorded by the slaughterhouse during 2012

and 2013 confirms that in autumn there was an increase of more than one kg with

respect to the summer (169.1 vs. 170.6 kg in 2012 and 169.4 vs. 170.9 kg in 2013).

This seasonal increase of live weight of heavy pigs was also shown by the national

slaughter statistics of heavy pigs [26].

The incidence of hemorrhages was greater (P < 0.05) in spring and in autumn while

the veining defect was greater in spring. These results cannot easily be attributed to

differences in seasonal conditions. The lowest occurrences of lacerations and

hemorrhages were found in summer. Warmer temperatures recorded during this

season probably lead to a more careful handling in order to reduce the risk of

61

transport mortality [15,25,27]. It is well known that high environmental

temperature is a risk factor for transport mortality [28,29].

A significant source of variation in the incidence of raw ham defects was found in

studies over several years. The higher incidence of all defects was found in 2013,

especially for hematomas and veining. Crisis-invested DPO dry-cured ham

production became more important in 2013 with respect to the previous year. It is

probably that the reduction of employees for reducing costs lead to an overworking

of staff involved in transport and in pre-slaughter handling with a consequent

reduction of its quality. The detrimental effect to animal welfare due to overworking

of employees involved in animal handling was highlighted by Grandin [30].

Table 4. Results of variance analysis and least-squares means of defects

incidence by sources of variation.

Sources of variation Hematomas (%) Lacerations (%) Hemorrhages (%) Veining (%) P / Least squares

mean P / Least squares

mean P / Least squares

mean P / Least squares

mean Distance 0.0079 <0.0001 <0.0001 <0.0001

1 (11–37 km) 4.57 a 1.48 b 1.32 b 11.04 a 2 (38–86 km) 4.44 ab 1.31 c 1.34 b 11.14 a 3 (89–170 km) 4.08 b 1.30 bc 1.08 c 9.24 b 4 (199–276 km) 4.78 a 1.91 a 1.77 a 11.50 a Pooled SEM(*) 0.11 0.06 0.05 0.22

Season <0.0001 <0.0001 <0.0001 <0.0001

Winter 6.53 a 1.32 b 1.19 b 12.05 b Spring 5.88 b 1.33 b 1.74 a 15.23 a Summer 3.58 c 1.28 b 0.96 c 8.70 c Autumn 2.87 d 2.13 a 1.71 a 8.18 c Pooled SEM(*) 0.12 0.06 0.05 0.24

Year <0.0001 0.0359 <0.0001 <0.0001

2012 3.70 b 1.42 b 1.24 b 9.73 b 2013 5.37 a 1.55 a 1.48 a 11.74 a Pooled SEM(*) 0.08 0.04 0.04 0.04

Distance × season 0.7421 0.3412 0.0352 0.5894 Distance × year 0.1526 0.7083 0.0137 0.0007

62

Season × year 0.1926 0.0056 <0.0001 <0.0001 (*) SEM: Standard Error of Mean; Means in the same column without the same letter differ

significantly (P < 0.05).

Interaction between distance and season (Table 5) affected the incidence of

hemorrhages (P < 0.05). The defect increased markedly when distance increased

above 170 km in spring and in autumn (P < 0.05). Differences between seasons were

observed for less than 37 km distance (Cluster 1), with summer showing the lowest

incidence of hemorrhages (P < 0.05). Above this distance, the lowest incidences of

the defect were found in summer and in winter (P < 0.05).

Table 5. Effect of interaction between distance and season on the incidence of hemorrhages.

Cluster (Distance) Pooled

SEM 1 (11–37 km) 2 (38–86 km) 3 (89–170 km) 4 (199–276 km)

Hemorrhages (%)

Winter 1.36 ab x 1.14 ab y 0.87 b y 1.49 a y 0.0931

Spring 1.55 b x 1.71 b x 1.58 b x 2.16 a x 0.1096

Summer 0.98 ab y 1.00 ab y 0.65 b y 1.33 a y 0.0912

Autumn 1.48 b x 1.67 b x 1.52 b x 2.26 a x 0.1527 Least square means within a row with different letters (a–b) differ (P < 0.05); Least square means within a column with different letters (x–y) differ (P < 0.05).

Interaction between distance and year (Table 6) affected the frequencies of

hemorrhages and veining. They increased both in 2012 and 2013 when distance

increased above 170 km. The lowest incidences of these defects (P < 0.05) were

found for cluster 3 (89–170 km) in 2012 only.

Table 6. Effect of interaction between distance and year on the incidence of

hemorrhages and veining defects.

Cluster (Distance) Pooled

SEM 1 (11–37 km) 2 (38–86 km) 3 (89–170 km) 4 (199–276 km)

Hemorrhages (%)

63

2012 1.32 a 1.27 a 0.91 b 1.57 a x 0.0688

2013 1.33 b 1.42 b 1.28 b 1.99 a y 0.0796

Veining (%)

2012 10.39 a x 10.63 a 7.63 b x 10.64 a x 0.2838

2013 11.73 y 11.68 11.17 y 12.42 y 0.3366 Least square means within a row with different letters (a–b) differ (P < 0.05); Least square

means within a column with different letters (x–y) differ (P < 0.05).

Interaction between season and year (Table 7) affected the incidences of

lacerations, hemorrhages and veining. No differences between years were observed

for lacerations but these differences appeared for hemorrhages in summer and

autumn and for veining in spring, summer and autumn. These results confirmed the

effects of the season on the incidences of raw ham defects. With regards to the

influence of the year, as stated before, its effect appears related to a reduction of

the quality of animal handling.

Table 7. Effect of interaction between season and year on the incidence of lacerations, hemorrhages and veining defects.

Season

Pooled SEM Winter Spring Summer Autumn

Lacerations (%)

2012 1.19 b 1.31 b 1.16 b 2.23 a 0.0824 2013 1.47 b 1.35 b 1.41 b 2.04 a 0.0873

Hemorrhages (%)

2012 1.28 b 1.71 a 0.71 c x 1.53 b x 0.0711 2013 1.11 b 1.76 a 1.30 b y 1.90 a y 0.0802

Veining (%)

2012 11.40 b 17.55 a x 6.72 c x 6.60 c x 0.3040 2013 12.73 a 13.06 a y 11.27 ab y 10.14 b y 0.3369

Least square means within a row with different letters (a–c) differ (P < 0.05); Least square means within a column with different letters (x–y) differ (P < 0.05).

64

4. Conclusions

The study has examined the effects of travel distances from 1 to 276 km, showing

that both short and long journeys may have adverse effects on fresh ham defects.

The higher incidence of defects associated with short transport distances may be

due to the lack of time to lie down and recovery from loading stress, but probably

also to hauliers working quickly to accomplish all planned transports for a given day.

Additionally, these results could be related to the lower quality of rural roads, which

represent the major part of the route in short journeys. Extending the transport

distance above 170 km results in an increased incidence of raw ham defects; the

incidence of defects reported herein was not proportional to the increase of

transport distance. This research highlights the need for Italian pig industry to

improve pre-slaughter handling in order to increase the quality of raw hams for DPO

production, reducing the economic losses related to inadequate practices before

slaughter and increasing the welfare of animals to make it as good as possible.

However, additional research is necessary to understand seasonal influences on

frequencies of defects on the fresh hams destined for the DPO process.

Acknowledgements

The authors wish to acknowledge the technical assistance of quality assurance staff

of the abattoir for data collected at the slaughterhouse. This research was

supported by Regione Lombardia, Programma di Sviluppo Rurale 2007–2013, Misura

124.

Thank you to authors that collaborated and helped with the preparation of this

manuscript. All authors contributed equally to this work.

65

Conflicts of Interest

The authors declare no conflict of interest.

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16. Prosciutto di Parma. Dossier 2013; Parma Quality Institute: Langhirano, Italy, 2014; pp. 5–38.

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CHAPTER 3

The effect of the stress immediately prior to stunning in different breeds

on pro-, macroglycogen, lactate and pork meat quality traits

Eduardo A. de Oliveira¹, Agnese Arduini², Leonardo N. Costa², Francesco Tassone²,

Stefania Dall’Olio²

1Estudante de doutorado do Programa de Pós Graduação em Ciências Veterinárias,

Universidade Federal do Paraná, Brazil.

2Dipartimento di Scienze e Tecnologie Agro-Alimentari, Università di Bologna, Italy.

Corresponding author: Prof. Dr. Leonardo Nanni Costa, DISTAL, Facoltà di Agraria,

Università di Bologna, viale Fanin, 46, Bologna (BO), Italy. Tel +39.051.2096513 – Fax

+39.051.2096516. E-mail: leonardo.nannicosta@unibo.it

Abstract

Muscular glycogen plays an important role on the extent of tissue pH decline,

following slaughter. Two glycogen fractions were examined—proglycogen and

macroglycogen—which are metabolized with different priority. Little is known about

the effects of pre-slaughter handling or differences between breeds on glycogen

fractions and pork meat quality traits. Therefore, the aim of the study was to

evaluate the levels of lactate, pro-, macro-, and total glycogen, and meat quality

traits in two different muscles across three different breeds of pigs, in the presence

or absence of physical, pre-slaughter stress. Twenty-eight pigs of the Italian Large

70

White, Italian Duroc, and Pietrain breeds were subjected to rough (RPH) or gentle

(GPH) pre-slaughter handling. RPH group pigs were subjected, before stunning, to a

fast driving, which was supported by the use of electric prods, whereas GPH group

pigs were driven slowly, without electric prods. Pre-slaughter handling showed no

significant effects across breeds (P>0.05) on the levels of proglycogen and

macroglycogen within the longissimus dorsi muscle; however, glycogen levels were

lower in pigs subjected to RPH. In semimembranosus muscle, the level of

macroglycogen was lower in RPH group pigs (P<0.01); handling groups showed no

significant effect on proglycogen levels (P>0.05). In longissimus dorsi muscle, the pH-

24, drip loss, cooking loss, total loss, and shear force were not affected by handling

(P>0.05). We conclude that physical stress, as a result of rough handling before

slaughter, has potential to reduce the quality of pork meat.

Key words: Breeds; Glycogen; Meat quality traits; Pigs.

Introduction

The store of muscular glycogen, at the point of slaughter, plays a decisive role on the

successive rate and extent of post mortem glycogen metabolism in pig muscles

(Pösö and Puolanne, 2005). Glycogen synthesis and storage research (Alonso et al.,

1995) has led to a renewed interest on the two constituent fractions, i.e.

proglycogen and macroglycogen, which had been identified about 50 years prior

(Wismer-Pedersen and Briskey, 1961), and more recently described in greater detail

(Lomako et al., 1993; Alonso et al., 1995). The proglycogen fraction is acid-insoluble,

has a molecular weight (MW) of approximately 400 kDa, and a low ratio of glucose

units to protein; the macroglycogen fraction is acid-soluble, has a MW of about 104

kDa, and a high ratio of carbohydrate to protein content (Lomako et al., 1993).

These two forms are metabolized with different priorities under aerobic and

anaerobic conditions (Asp et al., 1999; Graham et al., 2001).

71

Short-term stress, immediately before stunning for slaughter, increases the decline

rate of pH and temperature in early post mortem, and consequently has a

detrimental effect on pork meat quality traits (D’Souza et al., 1998; Henckel et al.,

2000; van der Wal et al., 1999). In this situation, the muscular glycogen store is

subjected to a rapid degradation, both in vivo and early post mortem (Henckel et al.,

2002), but the role of the glycogen fractions in accelerating post mortem glycolysis

and their relationships with pork meat quality are not well known.

The purpose of the present study was to investigate the effect of short-term,

physical stress prior to stunning on proglycogen, macroglycogen, and lactate levels,

and meat quality traits, by examining two muscles of pigs belonging to three

different breeds.

Material and methods

The experimental protocol complied with the rules approved by the Ethic

Committee of University of Bologna. The physical stress conditions applied before

stunning were carried out in accordance with the Council Regulation (EC) procedure

number 1099/2009 (European Council, 2009).

Animals and experimental design

A total of 28 pure bred, unrelated, castrated pigs were used; these included 10

Italian Large White (ILW), 10 Italian Duroc (IDU), and 8 Pietrain (PI) pigs. The

selection criteria included pigs that were clinically healthy and were non-carriers of

the recessive T allele (or n allele) of the g.1843C>T polymorphism of the ryanodine

receptor 1 (RYR1 or halothane) gene. Pigs were collected when at ~30 kg live weight,

from the experimental research farm of our Department. They were randomly

distributed amongst 8 pens; two pens per breed, with equal numbers of animals per

pen. Pigs were fed ad libitum for 115 days using a commercial feed pellet, containing

16% crude protein (0.8% lysine), and 14.1 MJ/kg digestible energy. All pigs had ad

72

libitum access to water via a nipple drinker. At the end of the 115 day growing

period, all pigs were slaughtered within a single day, at the same abattoir. Mean live

weights and the standard deviation were 114.0 ± 20.05 kg for ILW, 123.0 ± 12.57 kg

for IDU, and 115.2 ± 9.24 kg for PI. Subjects of each breed were allocated randomly

to two groups (5 ILW, 5 IDU, and 4 PI per group): gentle pre-slaughter handling

(GPH, or non-stressed) and rough pre-slaughter handling (RPH, or stressed).

Pre-slaughter condition

The farm was located 127 km from the abattoir; transport time was approximately 2

hours, and the pigs were slaughtered immediately upon their arrival. The physical

stress treatment for pigs of the RPH group began approximately 5 minutes before

slaughter. These pigs were subjected to a fast driving, consisting of a 25-m run,

supported by the use of electric prods, using shocks no longer than one second. Pigs

in the GPH group were driven slowly for the same distance, without the use of

electric prods. All animals were stunned by electronarcosis (V 220, Amp 1.3) before

slaughter.

Sampling and meat quality analyses

Tissue pH, 45 min after slaughter (pH-45), was determined in the muscles

longissimus dorsi (LD) at the last rib, and in the Semimembranosus (SM) on the left

side of carcass. After 24 hours at a temperature of 0–4°C, pH was measured again

(pH-24) in the LD muscle, in the same position. For practical reasons, the hams were

sectioned before 24 hours had passed, so it was not possible to obtain a pH-24

reading for SM. pH was measured using a pH-meter equipped with a glass electrode

(model 5232, Crison, Italy). In conjunction with the pH-45 measurement,

approximately 10 g of sample tissue from LD and SM was collected from each

animal, immediately frozen, and stored in liquid nitrogen for subsequent analysis of

proglycogen, and macroglycogen, according to Adamo and Graham (1998). After

73

slaughter, blood samples were collected and plasma samples were used to

determine the concentration of lactate (Kit Randox).

Instrumental colour, cooking loss, drip loss, total loss, and shear force were

evaluated in LD from the left side of the carcass. The objective CIE colour (L*a*b*

system) was measured at 24 and 72 hours after slaughtering (Minolta Camera Ltda.,

Japan). Drip loss was evaluated in 10 g meat samples, which were weighed and

suspended in inflated bags, at 0–4°C, for 48 hours. Thereafter, the samples were

weighed again. Drip loss was determined as the difference between final and initial

weight, and expressed as a percentage (Honikel, 1998). To measure cooking loss,

samples were cooked in a water bath until the sample temperature reached 75°C

(Honikel, 1998). Cooking loss was calculated as the difference between the sample

weight before and after cooking, expressed as a percentage. After cooking, the

samples were allowed to stand to reach room temperature (~15°C), then cut into 1 ×

1 × 2 m pieces, and placed in a Warner-Bratzler apparatus (model 1221, Instron),

perpendicular to the direction of the muscle fibres. Total loss was calculated as the

sum of drip loss and cooking loss, and expressed as a percentage of the initial weight

of the sample.

Statistical analyses

Data were analysed by the mixed-model procedure (PROC MIXED) of SAS (Version

9.2). The model included the fixed effects of pre-slaughter handling group (RPH or

GPH), breed (ILW, IDU, PI), and their interactions. Where the interactions were

significant, the means were compared by Tukey-Kramer test at a significance level of

P=0.05. Carcass weight was used as a covariate, but was not significant and removed

from the model.

74

Results and discussion

Based on molecular diagnostic test, all pigs were homozygous g.1843CC for RYR1. In

our samples, the initial pH values (pH-45) in both muscles were greater than 6.0; the

final pH levels (pH-24) in LD were higher than 5.3. This finding confirms that these

were not PSE meats (Adzitey and Nurul, 2011).

The effect of pre-slaughter handling, across breeds, on pH-45, pH-24, lactate, pro-,

macro-, and total glycogen are shown in Table 1. In LD and SM, pH-45 was

significantly lower in the RPH group than in the GPH group (P<0.05), in agreement

with previous studies (Dokmanovic et al., 2014; Peres et al., 2014; Hambrecht et al.,

2004; Henckeç et al., 2000; van Der Walt et al., 1999).

Lactate levels in LD were significantly higher in the RPH group than in the GPH group

(P<0.001), also in agreement with the results of previous studies (D'Souza et al.,

1998; Hambrecht et al., 2004; Hambrecht et al., 2005). Despite higher levels of

lactate recorded in the RPH group, we found no significant difference between final

pH levels in the LD muscle. This is probably related to the short intensity and

duration of physical efforts before slaughter; the same result was found in studies

that evaluated similar intensities of pre-slaughter stress (Dokmanovic et al., 2014;

Peres et al., 2014; Rabaste et al., 2007).

The lack of an observed stress effect on lactate levels in SM (P>0.05) may be due to

the lower activity in this muscle of glycogen debranching enzyme (GDE), which is

essential to control the rate of glycogenolysis and glycolysis (Kylä-Puhju et al., 2005).

The GDE activity in LD and SM muscles of pigs was studied by Yla¨-Ajo et al. (2007),

and they found lower activity of GDE in SM, which may be a limiting factor in lactate

production. The difference in GDE activity between these two muscles is due to

differences in cooling rates and metabolic speed (Kylä-Puhju et al., 2005).

75

Table 1. Effect of pre-slaughter handling and breed on pH, proglycogen,

macroglycogen, total glycogen and lactate.

Variable Pre-slaughter

handling, Pre-s. Breed Pre-s Breed Interaction R-MSE Gentled Rough ILW ID PI

Longissimus dorsi, LD pH-45 minutes 6.43a 6.27b 6.44 6.36 6.25 0.04 ns ns 0.21 Lactate, µmol/g 37.01b 50.89a 39.91 43.60 48.34 0.005 ns ns 12.04 Macroglycogen, µmol/g 11.23 8.01 9.10 11.79 7.97 ns ns ns 4.38

Proglycogen, µmol/g 67.53 61.89 65.79 67.82 60.51 ns ns ns 27.44 Total glycogen, µmol/g 78.76 69.90 74.90 79.62 68.48 ns ns ns 26.92 pH-24 hours 5.51 5.50 5.55 5.51 5.47 ns ns ns 0.11

Semimembranosus, SM pH-45 minutes 6.46a 6.29b 6.46 6.35 6.31 0.01 ns ns 0.17 Lactate, µmol/g 32.65 39.12 31.55 41.67 34.45 ns ns ns 10.37 Macroglycogen, µmol/g 16.71a 9.72b 16.76 12.02 10.87 0.001 ns ns 4.88

Proglycogen, µmol/g 75.72 66.01 82.27 64.63 65.69 ns ns ns 20.17 Total glycogen, µmol/g 91.37 76.80 99.03 76.65 76.56 ns ns 0.04 ° 22.94 ns, not significant. ILW, Italian Large White. ID, Italian Duroc. PI, Pietrain. R-MSE, Root mean square error.

°=Interaction effect are showed on Figure 1; ab Different letters in the same row denote significant (P<0.05)

differences.

In LD, for all breeds, significant effects of different stress treatments upon the levels

of pro-, macro-, and total glycogen were not observed (P>0.05). This is probably due

to the low motor action of this muscle (Young et al., 2009). In SM, which has the

higher motor action, the pigs of the RPH group showed a significantly lower content

of macroglycogen than the GPH group (P<0.01). Macroglycogen is a source of

energy in aerobic metabolism (Asp et al., 1999), and is degraded during aerobic,

low-intensity exercise (Essén-Gustavsson et al., 2005). It follows, that the increased

movement of animals subject to physical effort before slaughter is responsible for

the decrease in macroglycogen levels. The lack of a significant pre-slaughter

handling effect on proglycogen of both examined muscles (P>0.05) is expected,

because proglycogen is largely degraded anaerobically (Sterten et al., 2010; Yla¨-Ajo

et al., 2007; Rosenvold et al., 2003). In the present study, the intensity of physical

76

effort that RPH group pigs were encouraged to was not enough to significantly alter

the levels of lactate in SM muscle, between the two groups of pigs.

A significant difference in SM glycogen levels was observed between different

breeds and their administered stress treatments (P<0.05, Table 1). ILW pigs from the

GPH group had significantly higher total glycogen levels than IDU pigs from the RPH

group (Figure 1). Moreover, the total glycogen levels in these two groups were

similar to the others. The effect of stress upon pigs is influenced by their genetic

background (Terlouw, 2005), and the variation in glycogen content between breeds

is due to the type of muscle fibres (Terlouw and Rybarczyk, 2008; Thompson et al.,

2006).

In both muscles, the levels of pro-, macro-, and total glycogen tended to be lower in

animals subjected to RPH; little difference between breeds was observed within

these parameters. Furthermore, the effect of handling varied between the two

muscle types, being most evident in SM.

77

Figure 1. Interaction between pre-slaughter handling (RPH, Rouge. GPH, Gentle) and

breed. a) total glycogen of Semimembranosus. b) b* 24 hours value in Longissimus

dorsi. abc Different letters denote significant (P<0.05) differences.

The effect of pre-slaughter handling and breed on instrumental colour, drip loss,

cooking loss, total loss, and shear force, in LD are shown in Table 2. Except for b* at

24 hours, there was no observed effect of stress on carcass meat quality traits (drip

loss, cook loss, total loss and shear force; P>0.05); this agrees with the results of

Peres et al. (2014).

As initial pH and lactate levels effect pork colour (Peres et al., 2014), it is also

expected that stress will, too. However, in this study, breed was responsible for the

main differences in pork colour.

The L* value, measured 24 and 72 hours after slaughter, was higher in PI than in ILW

pigs (P<0.01); IDU showed values similar to both PI and ILW (P>0.05). The lack of a

significant pre-slaughter handling effect on L* values is due to similar final pH levels

in both the RPH and GPH groups (Table 1). Lower final pH values are responsible for

greater damage to proteins, due to the approach of the isoelectric point; this

increases the amount of free water and causes greater scattering of light and

consequently higher L* values (Brewer et al., 2001). At the final pH (pH-24), the L*

values observed (less than 60) do not indicate the occurrence of PSE meat (Adzitey

and Nurul, 2011).

When animals physically exert themselves, there is an expected increase in levels of

myoglobin (Lawrie and Ledward, 2012), and consequently, an increase in a* values.

This was not significantly observed in our study (P>0.05), nor in a similar study by

Dokmanović et al. (2014). However, the effect of pre-slaughter handling stress upon

b* values varied according to the breed. When measured 24 hours after slaughter,

the values in RPH groups were lower than those in GPH groups, for Italian Duroc (ID)

78

and Pietrain (PI). without effect of handling group in Large White (Figure 1). When

measured 72 hours after slaughter, no significant difference was observed in b*

values between handling groups, but the ILW breed showed significantly lower

values when compared to the 24 hour values (P<0.01). It seems that pigs subjected

to more intense physical exertions, tend to have lower b* values, exhibited as more

yellow colour of the meat in the RPH group. Similar results were found by

Hambercht et al. (2005), who studied the effect of long duration transport in pork

quality.

In general, handling stress had little influence upon parameters that indicate meat

quality; any influence observed was largely restricted to instrumental colour. These

results could be due to the low intensity and short duration of pre-slaughter stress

that the RPH group pigs were exposed to. In addition, measurements were taken

from a postural muscle (LD), which is less affected by stress and movement than

locomotor muscles like the SM (Correa et al., 2014). The results suggest that the

effect of pre-slaughter handling upon pork colour of LD varies by breed.

Table 2. Effect of pre-slaughter handling and breed on color, drip loss, cooking

loss, total loss and shear force in Longissimus dorsi.

Variable Pre-slaughter

handling, Pre-s. Breed Pre-s Breed Interaction R-MSE Gentled Rough ILW ID PI

L* 24 hours 54.31 53.27 51.10b 53.61ab 56.65a ns 0.001 ns 2.67 a* 24 hours 9.17 8.45 8.64 9.16 8.63 ns ns ns 1.93 b* 24 hours 7.14a 4.97b 5.17b 6.98a 6.00ab <0.001 0.003 0.033 ° 1.03 L* 72 hours 55.75 56.84 53.45b 56.41ab 59.00a ns 0.017 ns 3.74 a* 72 hours 4.15 3.40 3.64 4.15 3.53 ns ns ns 1.67 b* 72 hours 11.47 10.71 10.16b 11.71a 11.41a ns 0.004 ns 1.00 Drip loss, % 3.25 3.41 2.70 3.55 3.74 ns ns ns 1.14 Cooking loss, % 18.41 16.97 18.80 17.92 16.35 ns ns ns 3.57 Total loss, % 21.06 19.78 20.96 20.82 19.47 ns ns ns 4.03 Shear force, kgf 2.44 2.27 2.47 2.23 2.30 ns ns ns 0.34

79

ns, not significant. ILW, Italian Large White. ID, Italian Duroc. PI, Pietrain. R-MSE, Root mean square error.

°=Interaction effect are showed on Figure 1; ab Different letters in the same row denote significant (P<0.05)

differences.

Conclusions

The effects of pre-slaughter handling in pork meat quality indicators were limited to

instrumental colour. Additionally, rough handling affected initial pH values and

lactate levels within the muscles. The levels of pro-, macro-, and total glycogen also

tended to be lower in animals subjected to RPH. We conclude that RPH, in the

conditions studied, is both a stress factor and a welfare concern that has the

potential to negatively affect meat quality.

Acknowledgments

The first author thanks the National Counsel of Technological and Scientific

Development of Brazil (CNPq) for supporting his postgraduate through the program

Science without Borders.

80

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84

GENERAL CONCLUSIONS

In a context of increasing attention towards animal welfare and excellent

productions, this thesis focused on the examination of the impact of pre-slaughter

management on pig’s behavioral and physiological responses, as well as on pork

quality. This thesis evidences how stockpeople handling, the transport practices and

in general pre-slaughter stress conditions influence the welfare of animals before

death, the carcass appearence and the technological quality of meat.

The conclusion of the first work is that during unloading at slaughter plant of pigs

and the driving toward the stunning, the operators handling is the main cause of

lesions on carcass and on hams. Handlers’behaviour affected damages score due to

an excessive and harsh use of driving tools, that in the studied scenario is

represented exclusively by the rubber stick. The use of this device induced panic in

pigs which hutting against facilities and the disorder of agitated animals make the

handlers frustrated and more stimulated in using moving devices too frequently in

order to contain animals and to feed properly the speed chain.

Animals behaviour responses are at the same time an indicator of poor welfare and

a risk factor for skin bruises with a probable reduced carcass important traits quality

as ham, loin and shoulder. Risk assessment application resulted a good method to

monitor pre-slaughter practices in order to identify carcass damage causes.

The second work of the thesis underlines how travel conditions and distances

significanlty affect the presence of defects in ham causing the rejection by DPO

Parma Consortium. Too short and too long transportation distances both increase

the frequency of damages in raw hams, specifically heamatomas, lacerations,

microhaemorrhages and veining. The incidence of defects and carcass damages in

general is connected also to the season of transport, with a tendency to greater

85

heamatomas and lacerations in cooler months and major blood-splashed defects

(microhaemorrhages and veining) in warmer seasons.

The third and last work of this thesis in fact shows that pro- macro- and total

glycogen levels tend to be lower in precious muscles as longissimus dorsi and

semimembranosus following ante-mortem rough handling and strong physical effort

independently from stress breed prone. Even if in the study there wasn’t found

relevant changes in quality indicators as colour, drip and cooking loss and share

force, the influence also on pH and lactate confirm, as other studies, that the

handling manner is a real potential cause of worseing of quality meat degree as well

as of pigs welfare.

Further and deeper studies should be performed to better understand which are the

critical points of pre-slaughter period in order to eliminate the risk factors in

general. The risk assessment could be a good approch for this object because is a

non invasive method that give a final more complete view of the whole process. If

more applied in slaughter pig field could be standardazied and adopted by all pig

industry as a control routine practice with a general improvement of both the state

of pigs and the quality of carcass and its single traits.

Surely trainig programs for operators and better handling practices at the abattoir

are the base for an improvement of animals conditions before slaughter and limiting

carcass and meat downgrading for damages.

To guarantee the best travel conditions considering the lenght of journey and the

period of the year improves also animal welfare and reducing ham defects and the

economical losses due to their refusal by DPO Consortia.

86

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